Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor

ABSTRACT

Described herein is the self-assembly of amphiphilic peptides, i.e., peptides with alternating hydrophobic and hydrophilic residues, into macroscopic membranes. The membrane-forming peptides are greater than 12 amino acids in length, and preferably at least 16 amino acids, are complementary and are structurally compatible. Specifically, two peptides, (AEAEAKAK) 2  (ARARADAD) 2 , were shown to self-assemble into macroscopic membranes. Conditions under which the peptides self-assemble into macroscopic membranes and methods for producing the membranes are also described. The macroscopic membranes have several interesting properties: they are stable in aqueous solution, serum, and ethanol, are highly resistant to heat, alkaline and acidic pH, chemical denaturants, and proteolytic digestion, and are non-cytotoxic. The membranes are potentially useful in biomaterial applications such as slow-diffusion drug delivery systems, artificial skin, and separation matrices, and as experimental models for Alzheimer&#39;s disease and scrapie infection. The sequence of the peptide, EAK16, was derived from a putative Z-DNA binding protein from yeast, called zuotin. The cloning and characterization of the ZUO1 gene are also described.

RELATED APPLICATION

[0001] This application is a Continuation-in-Part of U.S. Ser. No.07/973,326, filed Dec. 28, 1992, which is incorporated herein byreference.

GOVERNMENT FUNDING

[0002] This work was supported by Grant No. NIH-5R37-CA04186 from theNational Institutes of Health and by Grant No. N00014-90-J-4075 from theOffice of Naval Research. The U.S. Government has certain rights in thisinvention.

BACKGROUND

[0003] Macroscopic membranes play an important role in many biologicalprocesses at both the cellular and organismic level. In addition,membranes are used in a number of medical, research, and industrialapplications. Physiologically compatible membranes would be especiallyvaluable for biomedical products. At present, the self-assembly ofpeptides into macroscopic membranes has not been reported.

SUMMARY OF THE INVENTION

[0004] The invention relates to the discovery that a new class ofbiomaterials derived from peptides related to the yeast DNA bindingprotein zuotin. The oligopeptides are generally stable in aqueoussolutions and self-assemble into large, extremely stable macroscopicstructures or matrices when exposed to physiological levels of salt. Thebiomaterials are visible to the naked eye when stained with a dye, CongoRed, and can form sheet-like or fibril structures which have hightensile strength. These materials are substantially resistant to changein pH, heat, and enzymatic proteolysis. The biomaterials can have afibrous microstructure with small pores as revealed by electronmicroscopy.

[0005] A small peptide termed EAK16 (AEAEAKAKAEAEAKAK, aa 310-325 of SEQID NO: 2) was discovered serendipitously to self-assemble into stablemacroscopic membranes and filaments in the presence of millimolarconcentrations of salt. This invention relates to the self-assembly ofpeptides into stable macroscopic membranes and filaments. Peptides whichform membranes are characterized as being amphiphilic, e.g., havingalternating hydrophobic and hydrophilic amino acid residues; greaterthan 12 amino acids, and preferably at least 16 amino acids;complementary and structurally compatible. Complementary refers to theability of the peptides to interact through ionized pairs and/orhydrogen bonds which form between their hydrophilic side-chains, andstructurally compatible refers to the ability of complementary peptidesto maintain a constant distance between their peptide backbones.Peptides having these properties participate in intermolecularinteractions which result in the formation and stabilization of β-sheetsat the secondary structure level and interwoven filaments at thetertiary structure level.

[0006] Both homogeneous and heterogeneous mixtures of peptidescharacterized by the above-mentioned properties can form stablemacroscopic membranes and filaments. Peptides which areself-complementary and self-compatible can form membranes in ahomogeneous mixture. Heterogeneous peptides, including those whichcannot form membranes in homogeneous solutions, which are complementaryand/or structurally compatible with each other can also self-assembleinto macroscopic membranes and filaments.

[0007] Peptides which can self-assemble into macroscopic membranes andfilaments, the conditions under which membrane and filament formationoccurs, and methods for producing the membranes and filaments aredescribed and included in this invention.

[0008] Macroscopic membranes and filaments formed of the peptide EAK16have been found to be stable in aqueous solution, in serum, and inethanol and are highly resistant to degradation by heat, alkaline andacidic pH (i.e., stable at pH 1.5-11), chemical denaturants (e.g.,guanidine-HCl, urea and sodium dodecyl sulfate), and proteases in vitro(e.g., trypsin, α-chymotrypsin, papain, protease K, and pronase). Themembranes and filaments have also been found to be non-cytotoxic. Themembranes are thin, transparent and resemble high density felt underhigh magnification. Being composed primarily of protein, the membranesand filaments can be digested and metabolized in animals and people.They have a simple composition, are permeable, and are easy andrelatively inexpensive to produce in large quantities. The membranes andfilaments can also be produced and stored in a sterile condition. Thus,the macroscopic membranes and filaments provided by this invention arepotentially useful as biomaterial for medical products, as vehicles forslow-diffusion drug delivery, as separation matrices, for supporting invitro cell attachment and growth, for supporting artificial tissue,e.g., for in vivo use, and for other uses requiring permeable andwater-insoluble material.

[0009] Furthermore, the salt-induced assembly of the peptides intoinsoluble and protease-resistant protein filaments with a β-sheetsecondary structure is similar in some respects to the formation of theneurofibrillary filaments and amyloid plaques associated withAlzheimer's disease and the formation of scrapie prion protein filamentsliver cirrhosis, kidney amyloidosis, and other protein confirmationaldiseases. The formation of the macroscopic membranes and filaments can,therefore, be useful as a model system, e.g. to study these pathologicalprocesses. For example, such a model system can be used to identifydrugs which inhibit filament formation and are thus useful for treatingAlzheimer's disease and scrapie infection.

[0010] Peptide EAK16 was derived from a region of a yeast protein,zuotin, which exhibits a high affinity for DNA in the left-handed zconformation. Zuotin was identified by a gel shift assay for Z-DNAbinding proteins developed by the Applicants. Applicants further clonedand sequenced the gene encoding zuotin. Characterization of zuotinrevealed that the protein is a potential substrate for several proteinkinases and identified a putative DNA-binding domain. This inventionalso includes all or biologically active portions of the zuotin proteinand DNA encoding zuotin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the amino acid sequence of zuotin (SEQ ID NO: 2) anda number of features of this protein.

[0012]FIGS. 2A, 2B and 2C are photographs of the macroscopic membranesat low magnification.

[0013]FIGS. 3a, 3 b, 3 c, 3 d, 3 e, 3 f, 3 g and 3 h are serialphotographs of membranes under scanning electron microscopy (SEM).

[0014]FIG. 4 is the typical β-sheet circular dichroism spectrum of theEAK16 peptide.

[0015] FIGS. 5A-5C show the hypothetical interactions between EAK16molecules in the membranes and the secondary and tertiary structuresresulting therefrom.

[0016]FIG. 6 illustrates calculation of the inter-peptide distance ofionized and hydrogen bonding amino acid pairs.

[0017]FIG. 7A illustrates the self-complementarity andself-compatibility of a peptide (SEQ ID NO: 19).

[0018]FIG. 7B illustrates staggering of interacting peptides.

[0019]FIG. 7C illustrates peptide interactions in a heterogeneousmixture (SEQ ID NO: 20 and 21).

[0020]FIGS. 8A and 8B show the stability of the β-sheet structure ofpeptide EAK16 in water at different peptide concentrations.

[0021]FIGS. 9A and 9B show the thermal stability of the β-sheetstructure of EAK16.

[0022]FIG. 10 shows the stability of the β-sheet structure of EAK16 atdifferent pH.

[0023]FIG. 11 shows the stability of the β-sheet structure of EAK16 inguanidine-HCl.

[0024]FIG. 12 shows A) the sequence of synthetic oligonucleotides (SEQID NO: 22) used to isolate ZUO1; B) the N-terminal sequence (SEQ ID NO:23) of E. coli-expressed zuotin; and C) the nucleotide sequence of ZUO1.

[0025]FIG. 13 shows A) and B) the similarity between two regions ofzuotin and other proteins and C) the relative positions of the regionsin the protein.

[0026]FIG. 14 is a restriction map of the wild type ZUO1 locus and theURA3::zuo1 disrupted locus.

DETAILED DESCRIPTION OF THE INVENTION

[0027] This invention is based on the serendipitous discovery that asmall synthetic peptide, EAK16 (AEAEAKAKAEAEAKAK; 310-325 of SEQ ID NO:2), self-assembles into macroscopic membranes and filaments in anaqueous solution containing a small amount of salt. The sequence ofEAK16 was originally found in a region of alternating hydrophobic andhydrophilic residues in a yeast protein called zuotin (heavilyunderlined in FIG. 1). Initial study of EAK16 revealed that it has aβ-sheet secondary structure having unusual stability under variousconditions. The structure of the EAK16 and its unusual stabilityresembles that of β-amyloid protein. When EAK16 was added to the mediumof cultured nerve cells in order to test for toxicity, the formation ofmacroscopic membranes was unexpectedly observed. Observation of aβ-sheet structure was surprising, since the sequence of EAK16 waspredicted to form an α-helix (Chou and Fasman, 1978). Described beloware the structure and properties of the membranes and filaments;peptides which are able to self-assemble into membranes and filaments;methods and conditions for producing the membranes and filaments; theZUO1 gene and encoded zuotin, from which the EAK16 peptide sequence wasderived; and uses of the macroscopic membranes and filaments.

[0028] Structure of the Macroscopic Membranes

[0029] The EAK16 peptide was observed to form a membranous structurewith the appearance of a piece of transparent, thin (about 10-20 μm)plastic membrane when viewed under 100× magnification by phase-contrastmicroscopy (FIG. 2). The membrane shown in FIG. 2A was formed inphosphate-buffered saline (PBS) and transferred to a glass slide. Thephotograph was taken with a Nomarski optical microscope at 100×magnification. The colorless membrane is isobuoyant; thus, the image isnot completely focused.

[0030]FIG. 2B shows a membrane formed by adding a stock solution ofEAK16 (1 mg/ml) into PBS in the presence of 10 μg/ml Congo Red. Themembrane is shown at 20× magnification; each scale unit is 1 mm.

[0031] In FIG. 2C, a portion of a well-defined membrane with layers isclearly visible. The dimensions of this particular membrane areapproximately 2×3 mm.

[0032] The structure could also be observed with the naked eye bystaining it bright red with Congo Red, a dye which preferentially stainsβ-sheet structures and is commonly used to visualize abnormal proteindeposition in tissues (Pears, 1960).

[0033] At low magnifications (50-100×), the structure looks like a flatmembrane. At high magnifications (30,000×) under a scanning electronmicroscope (SEM), structural details are revealed (FIG. 3). Themembranes in FIG. 3 were formed by adding EAK16 to PBS and prepared forSEM by incubating in 5% glutaraldehyde at 4° C. for 30 minutes, thendehydrated with ethanol and liquid CO₂. The photographs were taken atmagnifications of 400, 800, 1600, 3000, 6000, 10000, 20000, and 30000×(FIGS. 3A-H). SEM revealed that the membrane is made up of individualfilaments that are interwoven. The architecture of the structure appearsto resemble high density felt or cloth. The diameter of the filamentsare approximately 10-20 nm and the distance between the fibers areapproximately 50-80 nm.

[0034] The β-sheet secondary structure of the membranes was confirmed bycircular dichroism (CD) spectroscopy (FIG. 4). The EAK16 peptide wasdissolved in water (10 μM) and the CD spectrum was taken. A typicalβ-sheet CD spectrum with an absorbance minimum at 218 nm and a maximumat 195 nm was detected. The β-sheet secondary structure of EAK16 wassurprising, since a number of short peptides containing alanine,glutamic acid and lysine were previously reported to adopt stableα-helices in solution (Marqusee and Baldwin, 1987; Marqusee et al.,1989; Padmanabhan et al., 1991). The self-complementaryoligopeptide-based biomaterial can alsobe processed into fibers. Theliquid peptide dissolved in water can be injected into a salt solutionvia a needle to produce thread-like materials. Optionally, this materialcan be further processed into fibers or threads.

[0035] Length and Sequence of Membrane-Forming Peptides

[0036] The effect of length and sequence on membrane formation wasexamined using several peptides (Table 1 and Example 3). The sixteenamino acid peptide, EAK16, with the sequence (AEAEAKAK)₂ (310-325 of SEQID NO: 2), could undergo self-assembly, while a twelve amino acidpeptide, EAK12, with the sequence AEAKAEAEAKAK (SEQ ID NO: 24), was ableto associate to a much smaller extent and formed small and non-uniformpieces of membranous material. EAK8, AEAEAKAK (310-317 SEQ ID NO: 2),which has a single unit of the repeat, did not form membranes underidentical conditions. Another 16 amino acid amphiphilic peptide, RAD16,having the sequence (RARADADA)₂ (SEQ ID NO: 3), was found to formmacroscopic membranes. Its 8 amino acid counterpart, RAD8, did not formmacroscopic membranes. These results indicate that peptide length is animportant factor in the formation of macroscopic membranes. The peptidelength should be more than 12 amino acids and preferably at least 16residues. Very long peptides, e.g., of about 200 amino acids, mayencounter problems due to insolubility and intramolecular interactionswhich destabilize membrane formation. Furthermore, peptides with a largeamount of hydrophobic residues may have insolubility problems. Theoptimal lengths for membrane formation will probably vary with the aminoacid composition.

[0037] Four non-amphiphilic peptides of varying amino acid sequence weretested for membrane formation; these are β-amyloid (1-28) (SEQ ID NO:4), β-amyloid (23-35) (SEQ ID NO: 5), substance P (SEQ ID NO: 6), andspantide (SEQ ID NO: 7). None of these peptides produced macroscopicmembranes under the identical conditions used with EAK16 and RAD16(Table 1). These results indicate that the alternating hydrophobic andhydrophilic self-complementary sequence of the peptide is important tomembrane formation.

[0038] Consideration of these results leads to a molecular model inwhich intermolecular interactions between the peptides stabilize thesecondary and tertiary structures of the membranes. Due to thealternating hydrophobic and hydrophilic residues of EAK16, β-sheetsformed from EAK16 peptides can present a hydrophobic face and ahydrophilic face. The four glutamic acids of EAK16 have carboxylside-chain groups with a pKa of 4.4-4.6 and the four lysines have aminoside-chain groups with a pKa of 10.0-10.2. At neutral physological, theside-chains of the glutamic acids and lysines are negatively andpositively charged, respectively. FIG. 5A predicts interaction betweenthree molecules of EAK16 peptide representing three antiparallelβ-sheets. Two β-sheet layers are held together by hydrophobicinteractions of alanine side-chains facing each other and two by ionizedpair or salt bridge interactions between the charged lysines andglutamic acids facing each other. The structure can also be formed ofparallel β-sheets. The tertiary structure comprising many β-sheets canbe extended in the Z direction. In FIG. 5A, the peptides are staggered.The staggered arrangement allows extension of the structure in the Xdirection, along the peptide backbone.

[0039] A three-dimensional view is shown in FIG. 5B. Each rectanglerepresents the plane of a β-sheet. In the Y dimension are theconventional β-sheet interactions, i.e., hydrogen bonding between theamino and carboxyl groups of the peptide backbones. In the X dimension,staggered coupling of the peptides within the β-sheets contributes tostability along the peptide backbone. In the Z dimension, interactionsbetween β-sheets are stabilized by the extended ionized pair andhydrophobic interactions. The combination of these interactions arethought to result in the formation of β-barrel structures which may bethe filaments observed under high magnification. The stagger distancebetween coupled peptides would determine the length of the filaments.

[0040]FIG. 5C illustrates the increasing levels of structural complexityas the peptides self-assemble into membranes.

[0041] Peptides which can form ionized pairs between their hydrophilicside-chains are referred to herein as complementary. Complementary pairinteractions can also occur as a result of hydrogen bonding between thehydrophilic side-chains. Thus, Asn or Gln can function as hydrophilicamino acids in place of charged residues in membrane-forming peptides.Since ionized pair interactions are stronger than hydrogen bonds,peptides with acidic and/or basic amino acid side-chains would beexpected to form more stable membranes than peptides with hydrogenbonding side-chains.

[0042] An additional stabilization factor is that complementary peptidesmaintain a constant distance between the peptide backbones. Peptideswhich can maintain a constant distance upon pairing are referred toherein as structurally compatible. The interpeptide distance can becalculated for each ionized or hydrogen bonding pair by taking the sumof the number of unbranched atoms on the side-chains of each amino acidin the pair (FIG. 6). For example, lysine has 5 and glutamic acid has 4unbranched atoms on its side-chains, respectively. An intermolecularinteraction between two EAK16 peptides would involve ionized pairingbetween the lysine amino group and the glutamic acid carboxyl group. Theinterpeptide distance for a lysine-glutamic acid pair would be 5+4=9atoms. Since all the pairs in a EAK16-EAK16 interaction would beLys-Glu, the interpeptide distance would be constant at 9 atoms alongthe length of the peptides (FIG. 7A). Thus, the EAK16 peptide is,self-complementary and self-compatible, and homogeneous mixtures ofEAK16 form membranes.

[0043]FIG. 7A illustrates a convenient way to check whether two peptidemolecules are complementary and structurally compatible. Variouspossibilities for staggering the coupling of the peptides areillustrated in FIG. 7B.

[0044] Amphiphilic peptides which are greater than 12 amino acids long,self-complementary and self-compatible, are expected to self-assembleinto macroscopic membranes in homogeneous peptide solutions. Twoexamples, EAK16 and RAD16, have been demonstrated. Table 2 lists someother peptides which are predicted to form membranes in homogeneousmixtures. These examples illustrate some of the variety of amino acidarrangement and composition of membrane-forming peptides.

[0045] The criteria of amphilic sequence, length, complementarity andstructural compatibility apply to heterogeneous mixtures of peptides.Suppose that two different peptides are used to form the membranes:peptide A, VRVRVDVDVRVRVDVD (SEQ ID NO: 20), has Arg and Asp as thehydrophilic residues and peptide B, ADADAKAKADADAKAK (SEQ ID NO: 21),has Lys and Asp (FIG. 7C). Peptides A and B are complementary; the Argon A can form an ionized pair with the Asp on B and the Asp on A canform an ionized pair with the Lys on B. A calculation of theinterpeptide distances in such pairs (FIG. 6), however, shows that thetwo peptides are not structurally compatible. Using a conversion factorof 3 Å per atom, the difference in interpeptide distance between the twopairs would be 3 Å. Applicants estimate that a variation in interpeptidedistance of more than 3-4 Å would destabilize intermolecularinteractions leading to membrane formation. Thus, in a heterogeneousmixture of peptides A and B, membranes would likely form, but they wouldbe homogeneously composed of either peptide A or B.

[0046] Using this sort of calculation, it becomes evident that a peptidecontaining both Asn and Gln as hydrophilic residues will probably formmembranes in which the peptides are staggered and Asn-Gln (interpeptidedistance=7) pairs are formed, but not Asn-Asn (distance=6) and Gln-Gln(distance=8) pairs.

[0047] Examples of peptides which are self-complementary andself-compatible, and thus, expected to form membranes in homogeneousmixtures, can be summarized in the following formulas:

(Φ_(i)Ψ_(j)Φ_(k)Γ_(l))_(n)  (1)

[(ΦΨ)_(k)(ΦΓ)_(l))]_(n)  (2)

[0048] where Φ, Ψ, and Γ represent neutral, positively and negatively,charged amino acids, respectively, which determine the composition andsequence; i, j, k, and l are integers and denote variable numbers; ndenotes the numbers of repeating units which also determines the lengthof the oligopeptides.

[0049] The EAK16 polymer has alanine as its hydrophobic residue on oneside of the sheet and clusters of two glutamates followed by two lysineson the ionic side. We refer to this pattern of two positive chargesfollowed by two negative charges as Modulus 2. In this case, the formula(2) is applied where both k and l are 2. We could also imagine otherpeptides which have Modulus 1, i.e., alternations of positive andnegative charges on one side of the β-sheet described by the formula(1), where i, j, k and l are 1, or Modulus 3 in which there are clustersof three negatively charged residues followed by three positivelycharged residues, etc, where k and l are 3 in the formula 92). Thenature of the hydrophobic side chain on the other side of the sheet canalso be varied.

[0050] Membranes can also be formed of heterogeneous mixtures ofpeptides, each of which alone would not form membranes, if they arecomplementary and structurally compatible to each other. For example,mixtures of (Lys-Ala-Lys-Ala)₄ (SEQ ID NO: 25) and (Glu-Ala-Glu-Ala)₄(SEQ ID NO: 26) or of (Lys-Ala-Lys-Ala)₄ and (Ala-Asp-Ala-Asp)₄ (SEQ IDNO: 27) would be expected to form membranes, but not any of thesepeptides alone due to lack of complementarity.

[0051] Peptides, which are not perfectly complementary or structurallycompatible, can be thought of as containing mismatches analogous tomismatched base pairs in the hybridization of nucleic acids. Peptidescontaining mismatches can form membranes if the disruptive force of themismatched pair is dominated by the overall stability of theinterpeptide interaction. Functionally, such peptides can also beconsidered as complementary or structurally compatible. For example, amismatched amino acid pair may be tolerated if it is surrounded byseveral perfectly matched pairs on each side. Mismatched peptides can betested for ability to self-assemble into macroscopic membranes using themethods described herein.

[0052] In summary, peptides expected to form macroscopic membranes havealternating hydrophobic and hydrophilic amino acids, are more than 12amino acids and preferably at least 16 amino acids long, arecomplementary and structurally compatible. The amino acids can beselected from d-amino acids, l-amino acids, or combinations thereof. Thehydrophobic amino acids include Ala, Val, Ile, Met, Phe, Tyr, Trp, Ser,Thr and Gly. The hydrophilic amino acids can be basic amino acids, e.g.,Lys, Arg, His, Orn; acidic amino acids, e.g., Glu, Asp; or amino acidswhich form hydrogen bonds, e.g., Asn, Gln. Acidic and basic amino acidscan be clustered on a peptide, as in EAK16 and RAD16. The carboxyl andamino groups of the terminal residues can be protected or not protected.Membranes can be formed in a homogeneous mixture of self-complementaryand self-compatible peptides or in a heterogeneous mixture of peptideswhich are complementary and structurally compatible to each other.Peptides fitting the above criteria can self-assemble into macroscopicmembranes under suitable conditions (described below).

[0053] The term peptides, as used herein, includes polypeptides andoligopeptides. The peptides can be chemically synthesized or they can bepurified from natural and recombinant sources.

[0054] For example, the macroscopic membrane can be formed byself-assembly of a peptide having the sequence(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_(n) (SEQ ID NO: 2 (aa 310-325) or(Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala)_(n) (SEQ ID NO: 40 (aa 1-8)), where nis greater than or equal to 2.

[0055] Formation of the Macroscopic Membranes and Filaments

[0056] The novel self-assembly of EAK16 was initially observed in tissueculture medium (Dulbecco Modified Eagle's Medium, Gibco BRL,Gaithersburg, Md.) containing calf serum. Membranes can also form fromEAK16 in phosphate-buffered saline (PBS: 150 mM NaCl, 10 mM sodiumphosphate, pH 7.4). Macroscopic membranes do not form in water butappear after addition of sodium phosphate to a water-peptide solution toan approximate final concentration of 100 mg/ml. Thus, salt appears toplay an important role in the self-assembly process.

[0057] Various metal cations were tested for effectiveness at inducingmembrane formation from EAK16. The results indicate that monovalentmetal cations induce membrane formation, but divalent cations primarilyinduce unstructured aggregates. Some anions, acetate, Cl⁻, SO₄ ⁻², andPO₄ ⁻², and organic ions, NH₄ ⁺ and Tris-Cl, were also tested and werenot found to induce membrane formation.

[0058] The order of effectiveness of the monovalent cations appears tobe Li⁺>Na⁺>K⁺>Cs⁺. Cs⁺ produces the least amount of membranes and inaddition, yields nonmembranous precipitates. The effectiveness of themonovalent cations appears to correlate inversely with the crystal radiiof the ions: Li⁺ (0.6 Å), Na⁺ (0.95 Å), K⁺ (1.33 Å), and Cs⁺ (1.69 Å)(Pauling, 1960). A correlation is also seen with the hydrated radii ofthe ions: Li⁺ (3.4 Å), Na⁺ (2.76 Å), K⁺ (2.32 Å), and Cs⁺ (2.28 Å), andwith the order of enthalpies of the monovalent cations (Pauling, 1960).It is not known at present if the monovalent metal cations act as acatalyst or if they are incorporated into the membrane. The size of thefilaments (10-20 nm) and interfilament distance (50-80 nm) in themembranes formed from EAK16 suggest that hydrated ions may stabilize theintermolecular interaction.

[0059] Concentrations of monovalent metal cations (NaCl) as low as 5 mMand as high as 5 M have been found to induce membrane formation within afew minutes. Thus, membrane formation appears to be independent of saltconcentration over this wide range. Salt concentrations of less than 5mM may also induce membrane formation, but at a slower rate.

[0060] The initial concentration of the peptide is a significant factorin the size and thickness of the membrane formed. In general, the higherthe peptide concentration, the higher the extent of membrane formation.Membranes can form from initial peptide concentrations as low as 0.5 mMor 1 mg/ml. However, membranes formed at higher initial peptideconcentrations (about 10 mg/ml) are thicker and thus, likely to bestronger. Therefore, it is preferable when producing the membranes toadd peptide to a salt solution, rather than to add salt to a peptidesolution.

[0061] Formation of the membranes is very fast, on the order of a fewminutes, and seems to be irreversible (see below). The process isunaffected by pH≦12 (the peptides tend to precipitate out at pH above12), and by temperature. The membranes can form at temperatures in therange of 4 to 90° C.

[0062] Formation of the membranes is inhibited by the presence ofdivalent metal cations at concentrations equal to or greater than 100μM, which promote unstructured aggregation rather than membraneformation, and by sodium dodecyl sulfate (SDS) at a concentration of atleast 0.1%.

[0063] Properties of the Macroscopic Membranes

[0064] Once formed, the macroscopic membranes are stable in a variety ofaqueous solutions, including water, phosphate-buffered saline (PBS),tissue culture medium, serum, and also in ethanol, and can betransferred to and stored in any of these liquids. Membranes formed ofEAK16 and RAD16 have been found to be stable in water or PBS for atleast a week without any sign of deterioration. The membranes can betransferred from one solution to another using a solid support such as aspatula. They can be broken by cutting, tearing or shearing.

[0065] Membranes formed of EAK16 were found to be unusually stable undervarious conditions expected to disrupt them. Circular dichroism (CD)spectroscopy measurements further demonstrated the unusual stability ofthe β-sheet secondary structure of the peptide EAK16 (Example 4). Theβ-sheets can be thought of as the building blocks for the macroscopicmembrane structures and their unusual stability confirms the strength ofthe peptide interactions holding the membrane together.

[0066] The β-sheet structure of EAK16 was not significantly affected bydilution of the peptide, as seen in FIGS. 8A and 8B. FIG. 8A shows theCD spectra of EAK16 at 0.625, 1.25, 2.5, 5.0, 10, 15 and 20 μM peptideconcentrations in water. The Y axis is expressed as CD signal inmillidegrees in order to show the β-sheet stability of the peptide indiluted concentrations. These data show that, even at the lowestconcentration (0.625 μM), the characteristic β-sheet CD spectrum withminimum at 217 nm and maximum at 194 nm was still clearly recorded. TheCD signal is linearly proportional to the peptide concentration,suggesting that the β-sheet structure is stable in very diluteconcentrations. Note that the spectra cross an isosbectic point at 205nm, thus, indicating that the same structures exist at all the peptideconcentrations. FIG. 8B shows a plot of normalized peptideconcentrations from 0.625 to 20 μm vs. the mean residue ellipticity at218 nm. The stability of the β-sheet structure of EAK16 at very diluteconcentrations of the peptide contrasts with observations of otherβ-sheet forming peptides, such as β(29-42) and β(1-42) of the β-amyloidprotein (Barrow and Zagorski, 1991) and the TL-LRR1 peptide (23 residuelength) from the toll protein of Drosophila (Gay et al., 1991), whichshow a stable β-sheet only in high peptide concentrations.

[0067] The stability of the membranes was also tested under a range oftemperature, pH and chemical conditions. For these experiments,membranes were formed by adding 20 μl of a 0.5 mM stock solution ofEAK16 to 0.5 ml of PBS, and transferred into water or other solutions attest conditions.

[0068] The membranes were found to be stable in water over a wide rangeof temperatures: up to 95° and at 4°, −20°, and −80° C. The membranescould be frozen and thawed, although care was required in handling thefrozen membranes which were brittle. In boiling water, the membranestended to be sheared by the mechanical agitation. The CD spectra ofEAK16 were also found to be unaffected over the range of 25-90° C.(FIGS. 9A and 9B). FIG. 9B shows the CD spectra of EAK16 (8 μM) at 25,37, 55, 70 and 90° C. The thermal profile of EAK16 (FIG. 9A) shows a 22%decrease of mean residue ellipticity over this range. Such strongthermal stability of the defined β-sheet structure of EAK16 is unusualfor a small peptide. For example, these findings contrast with theα-helix-forming sixteen residue (Ala, Glu, Lys)-containing peptidesstudied previously (Marqusee and Baldwin, 1987).

[0069] The EAK16 membranes were also tested in water at pH 1.5, 3, 7 and11 at room temperature for at least a week and at 95° C. for about 4hours. The membranes were unaffected at these pH. The β-sheet structureof the peptide was also unaffected over this pH range; the pH profile(FIG. 10) shows a less than lot decrease of ellipticity. Precipitationof the peptide was observed at pH above 12.5. These findings suggestthat the overall β-sheet structure of EAK16 is not altered drasticallyin various pH even though charged residues would be neutralized undersuch conditions. It is possible that the complementary interactionsbetween the Glu and Lys side-chains are strong even when the carboxylgroups of the Glu residues have been protonated at pH 1.5 and 3, due totheir ability to form hydrogen bonds even when protonated.

[0070] The EAK16 membranes were further tested for resistance tochemical denaturation under the following conditions: 1% and 10% sodiumdodecyl sulfate (SDS); 1, 2, 3, 4, 5, 6 and 7 M guanidine-HCl; and 1, 2,3, 4, 5, 6, 7 and 8 M urea. The membranes remained stable in thepresence of these chemicals at room temperature for at least 4 days. Themembranes were also tested at 95° C. for 4 hours in 10% SDS; 7 Mguanidine-HCl; and at 8 M urea. At this temperature, the membranesdissolved in 8 M urea, but remained intact in SDS and 7 M guanidine-HCl.

[0071] CD spectra measurements also showed that the sheet structure ofEAK16 was not significantly affected by incubation in 0.1% SDS at 90°C., 1-7 M-guanidine-HCl at room temperature or 8 M urea at roomtemperature for over 16 hours. No significant differences in CD signalswere observed even under the strongest denaturing conditions of 7 Mguanidine-HCl (FIG. 11) and 8 M urea. This is surprising, since 7 Mguanidine-HCl and 8 M urea can effectively denature most proteins andproteinaceous aggregates, including other β-sheet forming peptides(Trudelle, 1975). Furthermore, these denaturants are expected todiminish hydrogen bonds and hydrophobic interactions. The stability ofthe EAK16 secondary structure suggests that the peptide moleculesstrongly interact to form an interwound β-sheet structure. This isconsistent with the interwound filamentous structures observed in thepresence of monovalent alkali salts.

[0072] The peculiar stability of the secondary and tertiary structure ofEAK16 observed may be explained by the additive stabilizing interactionsillustrated by FIG. 5. It is known from theoretical calculations thateach ionic bond can contribute approximately 5 Kcal/M and that theinteraction of apolar side-chains of alanines can contribute 1 Kcal/M.The hydrogen bonds between C═O and the N in the backbone of peptides canalso contribute approximately 3 Kcal/M. Therefore, EAK16 would have atotal interaction energy of about 72 Kcal/M per two molecules. Since theβ-sheets are staggered and overlayed, the additive interaction energy ismuch greater than that of individual molecules. The peptide bond itselfhas only 5-7 Kcal/M and is very stable. In addition, the hydrophobic andhydrophilic interactions between β-sheets seem to stabilize thesecondary structure under conditions in which β-sheets are usuallydisrupted. Thus, it appears that even under very harsh conditions, thestabilization energy of these membrane-forming peptides is greater thanthe disruption energy.

[0073] It is interesting to note that the EAK8 and EAK12 peptides do nothave such unusual stability. EAK8 exhibited a random coil structureunder identical conditions. EAK12 was found to denature from a β-sheetstructure at high temperatures, and to subsequently undergo reversiblehelix-coil transitions. This is consistent with our model, in whichstabilization in the direction of the peptide backbone (X dimension,FIG. 5) is also significant.

[0074] Further analysis by methods such as fiber X-ray diffraction andatomic force microscopy may provide more insights into the organizationand stabilization of the membrane structure.

[0075] The membranes and filaments also have some interesting and usefulbiological properties. They are highly resistant to digestion byproteases. Membranes and filaments formed from EAK16 were not degradedby trypsin, α-chymotrypsin, papain, protease K, or pronase at 100 μg/mlconcentration in the appropriate buffers when incubated at 37° C.overnight or at room temperature (25° C.) for a week, even though theEAK16 peptide contains potential protease cleavage sites.

[0076] The membranes also appear to be non-cytotoxic. The EAK16 peptideformed macroscopic membranes when added to a tissue culture of nervegrowth factor-differentiated rat PC12 cells. The peptides and resultantmembranes did not affect the appearance or rate of growth of the cells.

[0077] Uses of the Macroscopic Membranes and Filaments

[0078] The above-described macroscopic membranes and filaments haveseveral uses. Because they are stable in serum, resistant to proteolyticdigestion and alkaline and acidic pH, and are non-cytotoxic, thesemembranes and filaments are useful in biomaterial applications, such asmedical products (e.g., sutures), artificial skin or internal linings,slow-diffusion drug delivery systems supports for in vitro cell growthor culture and supports for artificial tissue for in vivo use. Themembranes can be made and stored in a sterile condition. For example,they can be produced using synthetic peptides and sterile PBS and storedin sterile PBS. The membranes can also be stored in a water/ethanolsolution. In addition, the membranes have a simple composition and canbe easily and relatively inexpensively produced in large quantities.They can be used in numerous applications in which permeable and waterinsoluble material are appropriate, such as separation matrices (e.g.,dialysis membranes, chromatographic columns).

[0079] Due to their permeability, the membranes described herein areuseful as slow-diffusion drug delivery vehicles for protein-type drugs,including erythropoietin, tissue type plasminogen activator, synthetichemoglobin and insulin. The drug could be wrapped in layers of membrane,which would permit slow release of the drug and may extend the half-lifeof the drug in the bloodstream. Because the membranes are resistant todegradation by proteases and stomach acid (pH 1.5), drug deliveryvehicles made of these membranes could be taken orally.

[0080] The extremely small pore size of the membranes may make themuseful as filters, for example, to remove virus and other microscopiccontaminants (see e.g., Erickson, 1992). The pore size (interfilamentdistance) and diameter of the filaments in the membranes can be variedby varying the length and sequence of the peptides used to form themembranes.

[0081] In view of the conductive nature of histidine, membranes andfilaments manufactured with the amino acid, histidine, will be useful asa conductive biopolymer.

[0082] Modification of the membranes may give them additionalproperties. For example, the membranes may be further strengthened bycross-linking the peptides after membrane formation by standard methods.Collagen may be combined with the peptides to produce membranes moresuitable for use as artificial skin; the collagen may be stabilized fromproteolytic digestion within the membrane. Furthermore, combiningphospholipids with the peptides may produce vesicles.

[0083] The membranes may also be useful for culturing cell monolayers.Cells prefer to adhere to non-uniform, charged surfaces. The chargedresidues and conformation of the proteinaceous membranes promote celladhesion and migration. The addition of growth factors, such asfibroblast growth factor, to the peptide membrane can further improveattachment, cell growth and neurite outgrowth.

[0084] Cells were observed to adhere to EAK16 membranes floating in thetissue culture dish. Cell attachment occurs through direct interactionswith the biopolymer membrane as shown by attachment in serum-free mediumand cyclohexamide treatment of cells. Cell attachment to biopolymermembranes is robust in Ca⁺⁺ and Mg⁺⁺ free, EDTA containing medium, whichindicates that the attachment phase is integrin independent.

[0085] Certain peptide polymers of this class contain sequences whichare similar to the cell attachment ligand RGD. The suitability of thesebiomaterials for supporting in vitro cell growth was tested byintroducing a variety of cultured primary and transformed cells tohomopolymer sheets of EAK16, RAD16, RADA16, and heteropolymers of RAD16and EAK16. The RAD-based peptides are of particular interest because thesimilarity of this sequence to RGD. The RAD sequence is a high affinityligand present in the extracellular matrix protein tenascin and isrecognized by integrin receptors.

[0086] In addition, the permeability of the membranes would permitdiffusion of small molecules, such as growth factors or peptidehormones, to the underside of cell monolayers, thus, presenting thepotential for tissue culture of differentiated cells and/or stratifiedcell layers.

[0087] Cells can be grown in culture and can be engineered to producevaluable products (e.g., growth factors, interferon). These systems areknown to offer many advantages over harvesting such products fromanimals. Many cells require adherence to a surface, such as tissueculture plastic, resulting in a surface-to-volume limitation. Thebiopolymer materials of the present invention, containing cells, can bestacked in a vessel containing culture medium, improving the density ofcells grown in this manner. The porous microstructure of the biopolymerscan also be useful for encapsulating cells. The pore size of themembrane can be large enough to allow the diffusion of cell products andnutrients. The cells are, generally, much larger than the pores and are,thus, contained.

[0088] A wide variety of cells can grow on these biopolymers. Thisindicates that artificial tissues can be grown in vitro using biopolymersubstrates. Evidence indicates that the EAK16 biopolymers are notantigenic (e.g., do not provoke an immune response). While otherpolymers based on organic chemicals and silicone have been used fortransplantation purposes, many of these materials can degrade intohazardous components. In contrast, the degradation of our oligopeptidebiopolymers yields amino acids, which are non-toxic and can be utilizedby tissues to make proteins.

[0089] The filamentous structure of the membranes and filamentsdescribed herein is similar to the structure of silk fibroin protein,which consists largely of glycine-alanine-serine or alanine-glutaminerepeats and forms stable β-sheet filaments, although the silk fibroinprotein has a molecular weight greater than 360,000 (Lizardi, 1979),whereas EAK16 has a molecular weight of only 1,760. The filaments formedby EAK16 are much finer than silk fibers. The membranes formed by EAK16and other amphiphilic peptides described herein can be useful for makingvery thin, transparent fabric.

[0090] In addition, it is interesting that the neurofibrillary tanglesand amyloid plaques associated with neuropathological conditions, suchas Alzheimer's disease, are salt-dependent aggregates of β-amyloidprotein with extremely stable and highly insoluble β-sheet structure(Iqbal and Wisniewski, 1983). The aggregated Alzheimer's filament has adiameter of approximately 10-15 nm (Hilbich et al., 1991; Iqbal andWisniewski, 1983; Halverson et al., 1990; Kirschner et al., 1987),similar to the dimensions of the EAK16 peptide filaments. Orderedfilamentous aggregates (approximately 7-10 nm in diameter) have alsobeen reported in another β-sheet forming peptide, TL-RR1, a 23 aminoacid peptide segment found in the Drosophila Toll protein (Gay et al.,1991). Moreover, the scrapie prion protein also stains with Congo Redand forms aggregated filaments which are extremely stable and resistantto proteases. Thus, the formation of the macroscopic membranes mayprovide a useful model system for investigating the properties ofbiological proteins structures with such unusual properties as extremeinsolubility and resistance to proteolytic digestion. Studies in such amodel system may provide insights into the pathology and potentialtreatment of conditions characterized by the presence of these proteinsor proteinaceous structures. For example, drugs which inhibit theself-assembly of the EAK16 peptide or other membrane-forming peptideinto filaments or filamentous membranes can be identified. Drugsidentified by such a method may be useful for treating Alzheimer'sdisease or scrapie infection.

[0091] The invention further relates to the regeneration of nerves withfilaments of the membranes of the present invention. Nerve regenerationcan be promoted and directed by transplanting filaments of RAD16 alongthe correct path to their targets. This would be extremely useful forpatients with severed peripheral nerves. Such a technology could berefined to help people with spinal cord injuries. Neurite outgrowth ofNGF differentiated PC12 cells, retonic acid-differentiated humanneuroblastoma and mouse cerebellum granule cells occurs on RAD16-basedbiopolymers, but not on EAK16-based biopolymers, indicating that thereis substrate specific support for such specialized functions.

[0092] In addition, the ability of small peptides such as EAK16 toself-assemble into membranes may be useful in origin of life studiesrelated to cell membranes and cellular compartmentalization. Apropos tothis sort of investigation is the interesting observation that the EAK16peptide shows partial nucleotide hydrolysis activity. This activity isprobably due to the ability of lysine and glutamic acid side-chains toperform general acid and base catalysis.

[0093] Zuotin

[0094] The sequence of EAK16 was originally found in a yeast proteincalled zuotin. Zuotin was identified by its ability to bindpreferentially to left-handed Z DNA in a gel shift assay developed byApplicants. The zuotin gene, ZUO1, was cloned and sequenced (FIG. 12;SEQ ID NO: 1). ZUO1 was found to encode a 433 amino acid protein havingseveral interesting features. In addition to the alternating alanine andcharged residues of the EAK16 sequence, the protein contains severalpotential phosphorylation sites (FIG. 1), including sites recognized bythe CDC28 (or cdc2) kinase (

), casein kinase II (◯), cAMP-dependent protein kinase (▾), tyrosinekinase (★), and protein kinase C (). Zuotin also contains a bipartitenuclear targeting sequence.

[0095] Two distinct regions of zuotin were found to be similar to knownproteins. One region (residues 111-165) was similar to E. coli DnaJ,yeast YDJ1, yeast SCJ1, yeast SIS1, SEC63 (or NLS1), avian polyomavirussmall t and large T antigens, Drosophila csp29 and csp32, and humanHDJ-1 (FIG. 13A). A second region (residues 300-363) of zuotin issimilar to several histone H1 variants, including some human, chickenand sea urchin variants (FIG. 13B).

[0096] Both partially purified yeast zuotin and bacterially expressedrecombinant zuotin exhibited a high affinity for DNA in the left-handedZ conformation. The region of zuotin from amino acids 306 to 339(heavily underlined in FIG. 1) is thought to be the DNA-binding domain.Mutational analysis showed that ZUO1 is not an essential gene, but thatdisruption of its function leads to slow cell growth.

[0097] The partial purification of the putative Z-DNA binding proteinfrom yeast S. cerevisiae, the cloning and characterization of its geneand functional analysis are further described in Example 5.

[0098] The following examples illustrate the invention further and morespecifically.

EXAMPLE 1 Peptide Synthesis, Purification, and Solubility

[0099] The peptides were synthesized by solid-phase peptide synthesis onan Applied Biosystems Model 430A peptide synthesizer coupler usingstandard N-tert-butyoxycarbonyl (t-Boc) chemistry and cycles usingn-methylpyrolidone (NMP) chemistry (Steward and Young, 1984). Both N-and C-termini of the peptide EAK16 were blocked to resemble its nativestate in the protein zuotin. The C-terminal amides were synthesized onp-methylbenzhydrylamine resin and the N-terminus of the peptide wasacetylated using acetic acid anhydride with an equivalent ofdiidopropylethylamine (DIEA) in dimethylformamide. The peptides werecleaved from the resin using hydrofluoric acid/anisole 10:1 (v/v)(Applied Biosystems, 1986).

[0100] The peptides were purified through HPLC (high pressure liquidchromatography) using a Vydac C₁₈ semi-preparative column, eluted with agradient of 5-60% acetonitrile in 0.1% trifluoroacetic acid (TFA), andlyophilized in a speed vacuum. Peptide purity was determined by analyticHPLC and the composition was determined by amino acid analysis.

[0101] EAK16 peptide stock solutions were prepared at a concentration ofapproximately 0.57 mM (1 mg/ml) in water. The molecular weight of EAK16is 1,760. EAK16 has a maximal solubility of 3 mM (about 5 mg/ml) inwater, but can be solubilized at up to 6 mM (about 10 mg/ml) in 23%actonitrile. The concentration was determined by the ninhydrin methodsusing internal controls.

EXAMPLE 2 Circular Dichroism Measurement

[0102] Circular dichroism (CD) spectra were taken on an Aviv Model 60DSspectropolarimeter using program 60HDS for data processing. BecauseEAK16 contains both positively and negatively charged residues, thepeptide itself can serve as a buffer. CD samples were prepared andmeasured at 25° C., unless otherwise indicated. All reagents wereultrapure and solutions were filtered through a 0.22 μM pore filterbefore use.

EXAMPLE 3 Preparation and Testing of Peptides for Membrane Formation

[0103] The EAK16, EAK12, and EAK8 peptides were synthesized by a peptidesynthesizer (Applied Biosystems) and purified by reverse phase HPLC andeluted by a linear gradient of 5-80% acetonitrile, 0.1% TFA. Theconcentration of the peptides was determined by dissolving dried peptidein solution (w/v) and centrifuging the solution. Then, a portion of thesolution was analyzed by hydrolysis with internal controls. The sequenceof the peptides were confirmed by microsequencing (Edman degradation)using the Applied Biosystems peptide sequencer (Steward and Young, 1984;Applied Biosystems, 1986). The compositions of the peptides wereconfirmed by hydrolytic analysis.

[0104] Substance P, Spantide, and β-amyloid (1-28) are available fromBachem. β-amyloid (1-28) was also described in Barrow and Zagorski(1991). Substance P, Spantide, and β-amyloid (25-35) were aminylated onthe C-terminal ends.

[0105] The EAK12 and EAK16 tested for membrane formation were acetylatedand aminylated at both N- and C-terminal ends. Blocking of both of theN- and C-termini of EAK16 appeared not to be essential for membraneformation. The peptides were initially dissolved in water (5 mg/ml) orin 23% acetonitrile (10 mg/ml). A volume of 5-10 microliters of thedissolved peptides were applied to the DMEM medium, PBS or water. Theformation of the membrane was first observed under a phase-contrastmicroscope and then, by the naked eye after staining with Congo Red.

EXAMPLE 4 Stability of the β-Sheet Structure of EAK16

[0106] Circular dichroism (CD) spectroscopy was used to monitor thestability of the β-sheet structure of EAK16 under various conditions.

[0107] Dilute peptide concentrations. EAK16 secondary structure wasfound to be stable in very dilute concentrations of the peptide. A 3 mMstock EAK16 solution was mixed in water to a concentration of 20 μM,allowed to equilibrate, and the CD spectrum measured. The solution wasthen diluted five times by two-fold serial dilutions to finalconcentrations of 15, 10, 5.0, 2.5, 1.25 and 0.625 μM, allowed toequilibrate and CD spectra taken. Reverse experiments, in which theconcentration of peptide was increased by adding more peptide, were alsodone and similar results were obtained.

[0108] Temperature. The CD spectra of 8 μM EAK16 at 25, 37, 55, 70 and90° C. in water were measured. The ratios of ellipticity at 194 nm/217nm remained approximately 4.0 over the temperature range of 25-90° C.(Table 3); such a high ratio suggests strong stability of the sheetstructure. The secondary structure of EAK16 was not substantiallyaltered over this range of temperature and an isosbectic point wasobserved at 202 nm. The thermal profile showed a 22% decrease of −[θ]₂₁₈nm deg. cm²/decimole (FIG. 9).

[0109] pH. The β-sheet structure of EAK16 was also not significantlyaffected by pH. The peptide consists of 4 positively charged lysine and4 negatively charged glutamic acid residues at neutral pH. Lysine has acalculated pKa of 10.0 and glutamic acid has a pKa of 4.4 in proteins.EAK16 has a calculated pI of 6.71. It was assumed that changes of pHwould have a great effect on the β-sheet structure, especially when thecharged groups are neutralized. However, CD spectra of EAK16 showed thatpH had little effect on the secondary structure over a pH range of 1.5to 11 (FIG. 10). The 3 mM stock solution of EAK16 was mixed with pHbuffers at pH 1.5, 3, 7, and 11 to a final concentration of 10 μM andallowed to equilibrate for 4 hours before taking CD measurements.Insignificant differences in ellipticity were observed at these pH. ThepH profile showed a less than lot decrease of −[θ]₂₁₈ nm deg.cm²/decimole from pH 1.5 to 3, 7 and 11 (FIG. 10). However, when pH wasincreased beyond 12.5, precipitation of the peptide was observed.

[0110] Chemical denaturants. CD spectra measurements also showed thatthe β-sheet structure of EAK16 was not significantly affected byincubation in SDS (1% at 90° C.), 7 M guanidine-HCl or 8 M urea for over16 hours. The peptide (3 mM stock) was mixed with water or differentconcentrations of guanidine-HCl or urea and allowed to incubateovernight before taking CD measurements. SDS was added to a peptidesolution to a final concentration of 0.1% and incubated for 30 minutes.No significant differences in CD signals were observed even under thestrongest denaturing conditions of 7 M guanidine-HCl (FIG. 11) and 8 Murea.

EXAMPLE 5 Identification, Cloning and Characterization of the YeastProtein Zuotin Introduction

[0111] DNA is capable of undergoing a number of conformational changes;the most dramatic of these is from right-handed B-DNA to left-handedZ-DNA. There are several conditions that are known to stabilize Z-DNA.For example, poly(dG-m⁵dC) converts readily to left-handed Z-DNA invitro in the presence of millimolar concentrations of divalent metalsand polyamines, as well as small peptides (Behe and Felsenfeld, 1981:Rich et al., 1984; Takeuchi et al., 1991). Certain DNA sequences,especially alternating purines and pyrimidines can adopt theZ-conformation in response to negative supercoiling (Peck et al., 1982).Inside the cell, negative supercoiling can be generated duringtranscription (Liu and Wang, 1987; Tsao et al., 1989). Furthermore, theequilibrium between B- and Z-DNA can be influenced by proteins thatpreferentially bind one of the two conformations (Lafer et al., 1985).

[0112] A number of studies suggests that Z-DNA may exist in vivo(Jaworski et al., 1987; Rahmouni and Wells, 1989; Wittig et al., 1989);however, the extent of its occurrence is yet to be determined. Z-DNA hasbeen implicated in some important biological processes, such as generalDNA recombination (Bullock et al., 1986; Treco and Arnheim, 1986; Blahoand Wells, 1987; Wahls et al., 1990), and both positive and negativetranscriptional regulation (Nordheim and Rich, 1983; Naylor and Clark,1990).

Results

[0113] Detection of a Poly(dG-m⁵dC) Binding Protein in S. cerevisiae

[0114] Two probes that can be stabilized in the Z-form were used todetect potential Z-DNA binding proteins. One is an ˜600 bp fragment of³²P-labelled poly(dG-m⁵dC) that is stabilized in the Z-DNA form bymillimolar concentrations of MgCl₂ (Behe and Felsenfeld, 1981); theother is an oligonucleotide, [³²P](dG-BR⁵dC)₂₂, that can be stabilizedby millimolar concentrations of MgCl₂ or μM concentrations of Co(NH₃)₆³⁺. Yeast whole cell extract, nuclear extracts, and phosphocellulosecolumn fractions of yeast nuclear extracts were assayed by a gelretardation assay using either of these ³²P-labelled DNA fragments as aprobe. The gel retardation assay was carried out with the probe in thepresence of 10 mM MgCl₂ and a 400-fold excess of sheared salmon spermB-DNA. Under these conditions, the polymer assumes the Z-DNAconformation. A Z-DNA-specific antibody was used for positive control. 1μl of each fraction of yeast nuclear extract obtained by salt elutionfrom the phosphocellulose column with 0.2-0.5 M potassium phosphate (pH7.4) was added to the assays.

[0115] A distinctive band shift was detected in assays of thephosphocellulose column fractions of yeast nuclear extracts. Both wholecell extracts and nuclear extracts produced a similar band shift. Thenuclear extract fractions that did not bind to B-form DNA (Winter andVarshavsky, 1989) showed significant gel retardation using the probe[³²P]poly(dG-m⁵dC) in the Z-DNA form even in the presence of a 400-foldmolar excess of sheared salmon sperm DNA. A similar band shift resultedfrom binding with polyclonal anti-Z-DNA antibody. The pooled fractions(FI) also showed binding activity to the oligonucleotide probe,[³²P](dG-Br⁵dC)₂₂.

[0116] In order to determine if these band shifts were the result ofauthentic Z-DNA binding, negatively supercoiled plasmids of pUC19 andpUC19(GC) were used as competitor DNAs in gel retardation competitionassays. In these assays, [³²P] (dG-Br⁵dC)₂₂ was incubated in thepresence of a 2000-fold excess of sheared salmon sperm DNA withadditions as follows: 1) no addition; 2) a monoclonal anti-Z-DNAantibody (mAb); 3) mAb plus additional 50 ng of negatively supercoiledplasmid pUC19 (without a Z-DNA insert); 4) mAb plus 25 ng of negativelysupercoiled pUC19(CG) (containing a Z-DNA segment); 5) fraction F1 yeastprotein; 6) F1 plus additional 50 ng pUC19; and 7) F1 plus pUC19(GC).pUC19(GC) contains a 14 bp (dG-dC)₇ insert that can adopt theZ-conformation upon negative supercoiling. pUC19(GC) was assayed for itsresistance to BssHII digestion (Vardimon and Rich, 1983; Azorin et al.,1984) to confirm the presence of the Z-DNA prior to the assay.

[0117] The results showed that monoclonal anti-Z-DNA antibody (Moller etal., 1982), used as a positive control, exhibited specific complexformation in the presence of the competing plasmid pUC19, but not in thepresence of supercoiled plasmid pUC19(GC), which contains Z-DNA. Similarbinding specificity was observed when a partially purified yeastfraction (FI) was used instead of the anti-Z-DNA antibody. However, thecomplex observed with the protein fraction was more heterogeneous thanthat seen with the antibody. The Z-DNA binding activity of fraction FIwas further purified using affinity chromatography to apoly(dG-m⁵dC)-agarose column (FII) followed by Superose 12 (FIII) andMono-S chromatography. The resultant active fraction (FIV) included aprominent 51 kDa protein that was still quite complex.

[0118] Identification of Zuotin by Southwestern Blotting

[0119] In order to identify the specific protein that interacts with theZ-DNA probe, a Southwestern blot was employed. Proteins in the Mono-Scolumn fractions were transferred from a SDS-polyacrylamide gel to anImmobulon P membrane and exposed to conditions that favor renaturation.Subsequently, the filter was incubated in the presence of[³²P]poly(dG-m⁵dC), stabilized in the Z-form by 15 mM MgCl₂ and a300-fold excess of B-DNA (sheared salmon sperm DNA). An autoradiogramwas made of the Southwestern blot and compared with a silver-stained gelof the Mono-S fractions.

[0120] The results showed that the poly(dG-m⁵dC) probe bound a singlepolypeptide of ˜51 kDa present in fractions 12 and 13, both of whichwere active in the band shift assay. There was also a weak signal infraction 14. Although the fractions were quite complex, only the 51 kDaprotein was detected by autoradiography. The putative Z-DNA bindingprotein was named zuotin (from the Chinese, zuo, meaning left).

[0121] Purification of Zuotin and Cloning of ZUO1

[0122] Approximately 5 μg of zuotin were gel-purified for amino acidcomposition analysis and N-terminal sequencing. The composition ofhydrolyzed zuotin was obtained from the purified yeast protein and fromthe E. coli expressed or recombinant zuotin. Both were gel-purified andsubjected to HCl hydrolysis, then, analyzed by HPLC with internalcontrols. The deduced composition of zuotin is derived from the DNAsequence of the open reading frame (ORF) of ZUO1. The amino acidcomposition of zuotin was 20.5% (Arg, Lys, and His), 18.5% (Glu andAsp), 14.1% (Thr, Ser, and Tyr) and 68.16% (Arg, Lys, Asp, Glu, Ser,Thr, Ala, and Leu). The amino acid composition of zuotin is shown inTable 4.

[0123] N-terminal sequencing yielded the following:MFSLPTLTSDI(E/D)V[EV](N)(H/S)(D), where [ ] and ( ) indicate moderateand low confidence assignments, respectively.

[0124] Degenerate 32mer oligonucleotides were designed by “reversetranslation” of the N-terminal sequence (FIG. 12). Alternativenucleotides were introduced at five positions in the oligonucleotidesequence. Nucleotides later determined to be mismatched are underlined.The oligonucleotides were used as a probe in Southern hybridizationanalysis of yeast genomic DNA. The Southern blot revealed a singlehybridizing 2.4 Kb HindIII fragment. A yeast genomic EMBL3A library wassubsequently screened and 14 clones isolated. Restriction mapping andSouthern hybridization using the 32mer oligonucleotides revealed thatone of the isolates contained a 2.4 Kb HindIII hybridizing fragment.This HindIII fragment was subcloned and the nucleotide sequencedetermined. The DNA sequence revealed an open reading frame (ORF), whosetranslated N-terminal sequence corresponded exactly to that of theN-terminal sequence determined from purified zuotin (FIG. 12B). Toobtain the entire coding sequence of the ORF, a 3.1 Kb BamHI-EcoRIfragment was subcloned into the pBluescript vector (Stratagene), and thenucleotide sequence (Seq. ID #1) was determined by the dideoxy chaintermination method. This sequence data is available from the EMBLsequence data bank under accession number X63612.

[0125] ZUO1 Encodes a 433 Residue Protein

[0126] The 3.1 Kb BamHI-EcoRI fragment contains the entire zuotin codingregion (1291-2590), as well as 5′ and 3′ non-transcribed regions (FIG.12; SEQ ID NO: 1). In FIG. 12, tracts of alternating (AT)_(n), A or T inthe 5′ nontranscribed region that could serve as regulatory sites areunderlined with single lines and labelled. A homopurine/pyrimidine tract(with one exception) in the coding region that can adopt an alternativeDNA conformation is underlined with double lines. The potentialpolyadenylation site at the 3′ end of the gene is indicated. There is along ORF encoding a 433 amino acid (aa) protein with a calculatedmolecular weight of 49 kDa. A second ORF in the same orientation withinZUO1 potentially encodes a 168 amino acid polypeptide. It remains to beseen if there is a translated product from this second reading frame.The 5′ region of the 3.1 Kb fragment also has another ORF containing 210codons, which encodes a yeast analogue of the E. coli biotin synthetasegene (bioB).

[0127] The 5′ non-transcribed region of ZUO1 contains three A/T-richsegments and two alternating AT segments that may act as regulatorydomains from transcription of the gene. There is also a purine-richtract in the coding region that could adopt a DNA conformation differentfrom conventional B-DNA (McCarthy and Heywood, 1987). The coding regioncomprises 1299 base pairs and the transcript of ZUO1 is ˜1.7 Kb. ZUO1 islocalized on yeast chromosome VII near ADE3.

[0128] Zuotin has several interesting features: it consists of 13%alanine, 20.6% positively charged residues (lysine, arginine andhistidine) and 18.5% negatively charged residues (aspartic acid andglutamic acid) (Table 4). It has a pI of 8.8. The charged residues areclustered at the C-terminal end and there is one segment with 12 chargedresidues in a row (FIG. 1; + and − indicate positively and negativelycharged amino acids, respectively). There are two continuous perfect andone imperfect octad tandem repeats of alternating alanine and chargedamino acids (lysine and glutamic acid) in the alanine/lysine andarginine rich region (heavy underlining).

[0129] Zuotin also contains several potential phosphorylation sites,including sequences recognized by protein kinase C (), casein kinase II(◯), cAMP-dependent protein kinase (▾), and tyrosine kinase (★), aspredicted by Prosite (FIG. 1) (Bairoch, 1991). There are also twopotential CDC28 (or cdc2) phosphorylation sites (KTPFVRR from 21-27 andKTPIP from 201-205 of SEQ ID NO: 2

) (Moreno and Nurse, 1990). It has a bipartite nuclear targetingsequence: KKKAKEAAKAAKKKNKR from 340-356 of SEQ ID NO: 2 (wavyunderlining; Robbins et al., 1991).

[0130] There are several regions that are predicted to form an α-helix(Chou and Fasman, 1978) and this includes the repeated octad segment(heavy underlining, FIG. 1). However, when a 16 residue peptide (EAK16)of the repeated segment was synthesized and examined by circulardichroism, a distinctive β-sheet structure was observed.

[0131] The zuotin protein is structurally similar to several knownproteins. It shares a region of sequence similarity (residues 111-165)with DnaJ protein (SEQ ID NO: 8), which is involved in DNA replicationof bacteriophages λ and P1 (Liberek et al., 1988) and with several otheryeast proteins: YDJ1 (SEQ ID NO: 12; Caplan and Douglas, 1991), SIS1(SEQ ID NO: 11; Luke et al., 1991), SCJ1 (SEQ ID NO: 9; Blumberg andSilver, 1991) and SEC63 (or NPL1) (SEQ ID NO: 14; Sadler et al., 1989)(FIG. 13A). All of these proteins include the hexapeptide motif, KYHPDK(highlighted in bold, FIG. 13A). This hexapeptide motif is also presentin both the small t and large T antigens of avian budgerigar fledglingdisease virus (SEQ ID NO: 13; Rott et al., 1988) and in the csp29 andcsp32 proteins expressed in the retina and brain of Drosophila (SEQ IDNO: 10; Zinsmaier et al., 1990). The consensus sequence shown in FIG.13A (SEQ ID NO: 28) was obtained by computer analysis using theGeneWorks version 2.0 (1991) program. This consensus means that at leastfive identical amino acids are aligned in a row. − indicates negativelycharged amino acids; Φ, nonpolar amino acids; and nonconserved aminoacids.

[0132] There is also sequence similarity between another region ofzuotin (residues 300-363) and several histone H1 variants, includinghuman H1a, H1b and H1c, chicken H1.11L and H1.11R (SEQ ID NO: 17), andsea urchin H1β and H1δ (SEQ ID NO: 16). The conserved region is in theextended C-terminal tail of histone H1, a region rich in alanine, lysineand arginine residues. For example, the sequences from 300-363 in zuotinand 146-205 in sea urchin histone H1 (SEQ ID NO: 16) are 64% similar and46% identical (FIG. 13B). Identity is shown by a vertical line,similarity, by : or ..

[0133]FIG. 13C shows the relative positions of these conserved regionsin zuotin. The DnaJ similar region is located from amino acids 111 to165 and the histone HI similar region is located from amino acids 300 to363 of zuotin.

[0134] Construction and Analysis of zuo1 Mutants

[0135] In order to generate an interrupted zuo1 allele, the 1.2 KbHindIII fragment containing the S. cerevisiae URA3 gene was inserted ata unique HindIII site of the ZUO1 coding region (FIG. 14). Wild type DNAhas a 3.1 Kb EcoRI-BamHI fragment, whereas, the disrupted mutant has a4.3 Kb fragment. The plasmid pZUO1::URA3 was then linearized and used totransform DM27, a diploid ura3 yeast strain. Diploid Ura⁺ transformants,expected to be heterozygous at the ZUO1 locus, were selected andconfirmed to harbor disruption at the ZUO1 locus. The heterozygousdiploid strains were subsequently sporulated and subjected to tetradanalysis. Tetrads yielded four viable colonies: two large and two smallcolonies, in which Ura⁺ phenotypes co-segregated with the small colonies(i.e., a slow growth phenotype). Southern blot analysis using DNA fromfour tetrads revealed that all clones with the slow growth phenotypeharbor the 1.2 Kb insertion. The results showed that the insertion ofURA3 at the ZUO1 locus produces a similar slow growth phenotype.

[0136] Expression of ZUO1 in E. coli

[0137] In order to verify that the cloned S. cerevisiae ZUO1 geneencodes the putative Z-DNA binding protein, ZUO1 was expressed in E.coli using a T7 expression system (Studier et al., 1990). ZUO1 wascloned in pET8c at the unique NcoI and BamHI sites. When the lacUV5promoter was induced with IPTG, a protein band with an apparentmolecular weight of ˜51 kDa was detected. This protein was not seen inthe cell extract from pET8C transformants induced with IPTG nor in thecell extract from uninduced pETZUO transformants. Analysis of theprotein composition and the N-terminal sequence of the purified and theE. coli expressed or recombinant zuotin are essentially the same,indicating that zuotin was expressed correctly.

[0138] In attempting to purify zuotin expressed in E. coli, Applicantsfound that the recombinant zuotin was sequestered in inclusion bodies.However, enough material was in solution so that crude cell extracts andpartially purified zuotin could be prepared and assayed by the bandshift assay. Recombinant zuotin was partially purified by chromatographyof crude cell extract from induced pETZUO1 transformants throughphosphocellulose. ³²P-labelled poly(dG-m5dC) in the Z-form was incubatedwith the crude cell extract or with partially purified zuotin in thepresence of sheared salmon sperm DNA, poly(dG-dC), or poly(dG-Br⁵dC).Assays were run on: 1) labelled probe alone; 2) crude cell extract frominduced pET8c transformants; 3) crude cell extract from uninducedpETZUO1 transformants; 4) crude cell extract from induced pETZUO1transformants; 5) partially purified recombinant zuotin; 6) partiallypurified yeast zuotin; 7)-10) partially purified recombinant zuotin with20-, 40-, 100-, and 200-fold excess of salmon sperm DNA; 11)-12)partially purified recombinant zuotin with 20- and 40-fold excess ofpoly(dG-dC); 13)-14) partially purified recombinant zuotin with 20- and40-fold excess of poly(dG-Br⁵dC); 15)-17) partially purified yeastzuotin with 20-, 40-, and 200-fold excess of salmon sperm DNA; 18)partially purified yeast zuotin with 40-fold excess of poly(dG-dC); 19)partially purified yeast zuotin with 40-fold excess of poly(dG-Br⁵dC);and 20) an anti-Z-antibody (as control).

[0139] These gel shift assays showed that partial purification throughphosphocellulose yielded a fraction with Z-DNA binding ability. Thestrongly shifted band bound to labelled poly(dG-m⁵dC) even in thepresence of a 40-fold excess of salmon sperm DNA. On adding a 40-foldexcess of poly(dG-dC), which can form Z-DNA under certain conditions, asomewhat weaker band was visible. Similar results were found with zuotinisolated from yeast. It is interesting that the band shift with yeastzuotin migrated slightly further towards the positive side of the gelthan the bacterially expressed zuotin. It is possible that thisdifference could be due to differences in phosphorylation or otherpost-translational modifications that were not carried out in E. coli.Furthermore, the yeast zuotin bound Z-DNA more tightly than thebacterially expressed zuotin. It is known, for example, thatphosphorylation modifies the DNA binding activity of the yeastcentromere binding protein CBF3 (Lechner and Carbon, 1991). Analysis ofthe zuotin sequence using the Prosite computer program (Bairoch, 1991)suggests that zuotin may be phosphorylated. Attempts are now being madeto express zuotin in other systems, including those which phosphorylateproteins.

[0140] DNA Binding Properties of Zuotin

[0141] Yeast protein fractions containing zuotin are able to bind bothpoly(dG-m⁵dC) and oligo(dG-BR⁵dC)₂₂ in the Z-form as well as negativelysupercoiled pUC19(GC) containing a Z-form segment in the presence ofcompetitor B-DNA. Since zuotin has not been purified to homogeneity, itis difficult to obtain a precise Z-DNA binding constant. The yeastzuotin is estimated to have a several hundred-fold enhanced affinity forZ-DNA relative to B-DNA under the experimental conditions used, whilethe E. coli expressed zuotin binds less tightly. It would not besurprising if proteins which interact with Z-DNA have binding motifsdifferent from the binding motifs of several proteins known to interactwith B-DNA. B-DNA has distinct major and minor grooves, and B-DNAbinding proteins tend to either anchor their binding motifs in the majorgrooves or lie along the minor grooves (reviewed in Seeman et al., 1976;Pabo and Sauer, 1984; Churchill and Travers, 1991). Such binding toB-DNA is relatively tight and, in some cases, very specific. On theother hand, Z-DNA does not have a distinct major groove nor a highlyaccessible minor groove. Thus, it is possible that a Z-DNA bindingprotein would not be able to anchor its binding motif to the regioncorresponding to the major groove. It is possible that the bindingconstants of Z-DNA binding proteins are lower than those of B-DNAproteins. It has been shown that many DNA sequences can adopt theleft-handed Z-conformation (Rich et al., 1984). Thus, proteins thatrecognize Z-DNA may be conformationally specific as well as sequencespecific.

[0142] The binding of both yeast zuotin and bacterially expressed zuotinto poly(dG-m⁵dC) cannot be competed by 40-fold excess of poly(dG-dC),200-fold excess of poly(dA-dT), nor several thousand-fold of salmonsperm DNA. However, a mere 4-fold excess of poly(dG-Br⁵dC) in the Z-formcompletely inhibits zuotin binding to the probe. These results suggestthat zuotin may recognize the conformation of DNA rather than specificsequences per se. Another example of a protein that recognizes DNAconformation specifically is HMG1 (high mobility group protein). HMG1and proteins containing HMG1 domains bind DNA not by its sequence butrather by the DNA conformation at the crossing of two duplexes (Bianchiet al., 1992; Lilley, 1992).

[0143] The Biological Function of Zuotin

[0144] Previous studies have suggested that potential Z-formingsequences, i.e., (GC/GC)n, (GT/AC)n and other alternatingpurine/pyrimidine segments, exist in the intergenic regions of manyorganisms, including those of yeast (Hamada et al., 1982). The GT/ACsegments have been implicated in inducing homologous DNA recombinationin vivo (Bullock et al., 1986; Treco and Arnheim, 1986; Wahls et al.,1990). Also, a DNA strand transferase from human cells has beenpartially purified using a Z-DNA affinity column (Fishel et al., 1988).Recently, it has been shown that specific alternating purine/pyrimidinesegments in the upstream region of c-myc form Z-DNA during activetranscription.

[0145] The precise biological function of zuotin is not known at thepresent time. Since zuotin appears to be of nuclear origin, binds toDNA, is relatively abundant and may potentially be phosphorylated byprotein kinases and dephosphorylated by phosphatases during the cellcycle, it could be involved in chromosome organization. The threoninewithin the KTPFVRR and KTPIP sequences may be phosphorylated by the S.cerevisiae CDC28-CLN complex during the cell cycle.

[0146] Computer sequence comparison analysis revealed that two differentregions of zuotin have similarities with known proteins. The firstregion of zuotin (residues 111-165) has 46% identity and an overall 70%similarity with the N-terminus of E. coli DnaJ protein (residues 16-67)(SEQ ID NO: 8; FIG. 13A). The DnaJ protein is a heat shock proteininvolved in protein folding; it is also active in phage λ and P1replication in vivo and in vitro through its interaction with DnaBhelicase (Liberek et al., 1988; Zylicz et al., 1989). This region ofzuotin also shares similarity with several other yeast proteins: YDJ1 (ayeast DnaJ homolog), which may be involved in protein assembly into theendoplasmic reticulum and nucleus (SEQ ID NO: 12; Caplan and Douglas,1991); SCJ1, which is involved in protein sorting (SEQ ID NO: 9;Blumberg and Silver, 1991); and SIS1, which is an essential protein andmay be involved in yeast DNA replication by mediating a specificprotein-protein interaction (SEQ ID NO: 11; Luke et al., 1991).Similarity was also found to the yeast protein SEC63 (or NLS1), which isimportant for protein assembly into the endoplasmic reticulum and thenucleus (SEQ ID NO: 14; Sadler et al., 1989). Both YDJ1 and SIS1 haveseveral cysteines that could potentially form a zinc finger DNA bindingmotif, but zuotin has only one cysteine and no zinc finger motif couldbe found. All the above proteins have a conserved hexapeptide, KYHPDK,except SEC63, in which F has replaced Y (FIG. 13A). This peptide motifmay play an essential role in these diverse proteins. Moreover, bothsmall t and large T antigens (SEQ ID NO: 13) of the avian polyomavirus,budgerigar fledgling disease virus, as well as the csp29 and csp32 (SEQID NO: 10) proteins expressed in Drosophila retina and brain have thisidentical hexapeptide motif (FIG. 13A) (Zinsmaier et al., 1990).Recently, a human nuclear protein HDJ-1 has also been shown to besimilar at both the N- and C-termini to the DnaJ protein (SEQ ID NO: 15;Raabe and Manley, 1991).

[0147] A second region of similarity in zuotin (residues 300-363) isrelated to histone H1 and some of its variants, such as human H1a, H1band H1c, chicken H1.11L and H1.11R (SEQ ID NO: 17), and sea urchin H1βand H1δ (SEQ ID NO: 16) (FIG. 13B). It is significant that histones H2A,H2B, H3 and H4 do not have regions similar to zuotin. Calf thymushistone H1 has been shown to have a higher affinity to Z-DNA than toB-DNA and it is able to convert Z-DNA to B-DNA, a transition that can bemeasured using circular dichroism spectroscopy (Russell et al., 1983;Mura and Stollar, 1984). Also, the Drosophila histone H1 has beenpreviously purified using a Z-DNA affinity column and Z-DNA bindingassays. It is possible that zuotin has some elements of histoneH1-related activity in yeast.

[0148] A subportion of this histone-like region from amino acids 306 to339 (heavily underlined in FIG. 1) is a likely candidate for aDNA-binding domain. In related studies, Applicants found that thepeptides KAKAK (SEQ ID NO: 29) and KAK were able to bind to B-DNA andconvert it to the Z conformation. Amino acid substitutions at the middlelysine of KAKAK (SEQ ID NO: 30) resulted in a loss of activity, butchanges at the carboxyl-terminal K did not significantly affectactivity. A peptide, KAHAK, was active in converting B-DNA to Z-DNA onlywhen the histidine was protonated (at low pH). The KAKAX motif (SEQ IDNO: 31) (where X is variable) occurs twice in the 306-339 region andalso occurs in the peptide, EAK16. These observations are consistentwith the structural similarity of this region to histone H1.

[0149] In an attempt to see whether zuotin is found in other organisms,a Southern “zooblot” was carried out in which various DNAs were probedwith zuotin DNA. Of 12 plant and animal species that were probed underlow stringency, all were negative except yeast. This suggests thatzuotin is a yeast-specific protein.

[0150] Mutant yeast cells in which ZUO1 was disrupted exhibit a slowgrowth phenotype. Thus, the function of ZUO1 appears not to beessential, rather it may be involved in some activity that is needed tomaintain rapid cell growth.

MATERIALS AND METHODS

[0151] Yeast Strains and Media

[0152] The genotype and sources of yeast S. cerevisiae used in this workare as follows: DB2670, MATα, his3-Δ200, ura3-52, can 1, pep4::HIS3,prb1-Δ1.6R was obtained from D. Botstein; 20B-12, MATα, pep4-3, trp1 hasbeen previously described (Jones, 1977); DM27, MATα/α, his3/HIS3,leu2/LEU2, ade2/ADE2 ura3/ura3, trp1/trp1, cyh/CYH was obtained from D.Dawson. Cells were grown in YPD medium (1% yeast extract, 2%bactopeptone and 2% glucose). SD medium contained 0.6% Difco yeastnitrogen base without amino acids and 2% glucose. Nutrients essentialfor auxotropic strains were supplied at concentrations recommended bySherman et al. (1986). The plasmid, pUC19(GC), was obtained from B.Johnston. Characterization of the anti-Z-DNA Z-22 monoclonal antibodyand polyclonal goat anti-Z-DNA antibody have been described (Moller etal., 1982).

[0153] Preparation of the Poly(dG-m⁵dC) Affinity Matrix

[0154] Poly(dG-m⁵dC) DNA (Pharmacia) (1.6 mg in 3 ml) was digested to anaverage size of ˜600 bp using DNase I in 50 mM Tris-HCl (pH 7.5), 30μg/ml BSA in the presence of 2 mM MnCl₂ to produce blunt ends (Maniatiset al., 1982). The digested DNA was deproteinized and resuspended in T4DNA polymerase buffer in the absence of dNTP and incubated with T4 DNApolymerase at 10° C. for 10 minutes. Subsequently dGTP and biotinylateddCTP (ENZO) were added to 1 mM final concentration, and incubation wascontinued at 37° C. for 2 hours. DNA was then separated fromunincorporated nucleotides by phenol and chloroform extraction, followedby two ethanol precipitations. Then, DNA was dissolved in 0.1 M NaCl, 1mM EDTA, 10 mM Tris-HCl (pH 7.5) and incubated with 1 ml ofstreptavidin-agarose (BRL) overnight by gentle inversion. Under theseconditions, more than 60% of the input DNA was bound tostreptavidin-agarose as determined by A₂₆₀ measurement after pelletingthe agarose. The DNA matrix was then washed extensively with 40 columnvolumes of buffer (10 mM Tris-HCl, pH.7.5, 50 mM KCl and 15 mM MgCl₂).The column wash was assayed for unbound DNA (using the BluGenenon-radioactive nucleic acid detection system, BRL) to assess the columnstability.

[0155] Purification and Sequencing of Zuotin

[0156] The preparation of crude nuclear fractions has been describedpreviously (Winter and Varshavsky, 1989). Crude total cell extract (fromstrain DB2670) was prepared from mid-log phase yeast cells (18 L at˜2.4×10⁷ cells/ml). Yeast cells were collected at 4500×g for 10 minutesat 4° C. and washed twice with water. The cell pellet was resuspended in600 ml of 0.2 M potassium phosphate (pH 7.5), 5 mM EDTA, 1 mMphenylmethylsulfonyl fluoride (PMSF), 0.8 μg/ml pepstatin A and 10%glycerol. The final volume was ˜800 ml. The cell suspension wasprocessed through a high pressure compressor at 900 psi ˜50 passages tobreak the cells. The suspension was then centrifuged at 12,000×g for 10minutes at 4° C. The supernatant (˜720 ml) was frozen at −80° C.Fractionation of whole cell extract on a phosphocellulose P-II columnand on a Mono-S column were as described by Winter and Varshavsky(1989). The pooled fraction (FI) containing binding activity topoly(dG-m⁵dC) in the presence of sheared salmon sperm DNA was diluted6-fold to achieve a potassium phosphate concentration of ˜50 mM (420ml). MgCl₂ was then added to a final concentration of 15 mM for loadingon the poly(dG-m⁵dC) affinity column. The column was washed with 10volumes of the column buffer and eluted with a linear gradient of 25 ml0.1-1.0 M KCl without MgCl₂ (FII).

[0157] The eluted proteins were then loaded on a Superose-12 gelfiltration column. One ml fractions were collected and assayed for theZ-DNA binding activity (FIII). These fractions were pooled, diluted3-fold and loaded on a Mono-S column. The Mono-S column was washedextensively and eluted with a linear 47 ml gradient of buffer, 0-1.0 MNaCl, in 10 mM sodium phosphate (pH 7.2). The 1 ml fractions containingZ-DNA binding activity (FIV) were analysed by SDS-PAGE.

[0158] For protein composition and sequence analysis, the pooled Mono-Scolumn fractions 12 and 13 were resolved on a 9% polyacrylamide-SDS gel.After electrophoresis, the protein was electroblotted on Immobulon(Millipore), briefly stained with Coomassie Blue and washed. The bandwith an apparent molecular weight of 50 kDA was excised and ¼ of thesample was used for amino acid composition analysis. The remainingsample was N-terminal sequenced by automated Edman degradation in anApplied Biosystems 470A Protein Sequencer equipped with on-line 120A PTHanalyser.

[0159] Band Shift and Competition Experiments

[0160] A gel retardation assay was employed to detect proteins withaffinity to left-handed Z-DNA. In these assays, two kinds of left-handedDNA probes were used. One was poly(dG-m⁵dC) (Pharmacia) stabilized inthe left-handed Z-form by 15 mM MgCl₂. The DNA probe was made asfollows: DNA polymer was digested with DNase I in the presence of 2 mMMnCl₂ and fragment of ˜600-1000 bp were gel purified and labelled withT4 DNA polymerase in the presence of dCTP and [α-³²P]dGTP (3000 Ci/mM)(Maniatis et al., 1982). The other probe was derived from a synthetic44mer oligo(dG-Br⁵dC)₂₂, labelled using Klenow polymerase with[α-³²P]dGTP, and stabilized in the Z-DNA conformation by 10 mM MgCl₂, or0.1 mM Co(NH₃)₆ ³⁺. In the assay reaction, 2 μl of diluted fractionsamples were incubated for 20 minutes at room temperature in 20 μl of 50mM Tris-HCl (pH 8.0), 15 mM MgCl₂, 5% sucrose, 0.1% Triton X-100, 10 mMβ-mercaptoethanol, a 2000-fold excess of sheared salmon sperm DNA, andthe labelled Z-DNA probe. In the competition assays, supercoiled pUC19and pUC19(CG) were added to the reaction samples separately. The sampleswere then electrophoresed in 1.5% agarose for poly(dG-m⁵dC) (10 mMMgCl₂, 1×TBE, pH 8.4) or 4% polyacrylamide for (dG-Br⁵dC)₂₂ (1×TBE, pH8.4). After electrophoresis, the gel was dried and exposed to X-rayfilm.

[0161] Southwestern Blotting

[0162] Mono-S column fractions containing proteins were electrophoresedon 9% polyacrylamide-SDS gel, as described by Laemmli (1970). Afterelectrophoresis, the gel was soaked in the running buffer without SDSbut with 20% methanol at room temperature for 45 minutes with agitation.The proteins were then blotted onto two sheets of Immobulon P membrane(Millipore) at 1.5 mA/cm² at room temperature for 60 minutes. Aftertransfer, the membrane was washed once with 5% milk powder, 30 mM HEPES(pH 7.4) (Celenza and Carlson, 1986); then, washed three times (3minutes each) with 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 10 mM MgCl₂(STEM). After washing, the membrane was incubated with 15 ml of theabove buffer containing 10 μg/ml sheared salmon sperm DNA. A DNA probe(average size 1300 bp) (at 2.55×10⁶ c.p.m./ml of poly(dG-m⁵dC)) wasadded to a final concentration of 30 ng/ml and the incubation continuedfor 1 hour at room temperature with gentle agitation (50 r.p.m.). Inthis buffer with 10 mM MgCl₂, the polymer is in Z-DNA conformation (Beheand Felsenfield, 1981). The membrane was then washed four times (8minutes each) with STEM at room temperature. The membrane was air-driedand exposed to X-ray film.

[0163] Cloning and Sequencing of ZUO1

[0164] The first 11 amino acids of the N-terminal sequence of zuotin(MFSLPTLTDI) were used to design oligonucleotides, with yeast codonusage as a guideline (Sharp et al., 1986). The pools contained an equalmolar mixture of 64 different 32mer sequences as follows: (SEQ ID NO:22) 5′-ATGTTTTCTTTGCCAACTTTGACTTCTGATAT-3′.           C     T  C     C     C (FIG. 12A)

[0165] These oligonucleotides were gel-purified and labelled using[γ-³²P]ATP and T4 polynucleotide kinase to a specific activity 200-500μCi/μg. The labelled oligo-nucleotides were first used in genomicSouthern blot hybridization to determine optimal conditions forscreening a phage λ library of yeast DNA. For this, yeast DNA wasdigested with HindIII, separated on an 1% agarose gel and blotted onto aGene Screen filter (New England Nuclear). The filter was hybridized at40° C. in 4.5% SDS, 0.34 M NaCl, 1 mM Na-EDTA, 10 mg/ml BSA and 0.16 Msodium phosphate buffer (pH 7.0) overnight. The filters were then washedat temperatures between 40 and 80° C. with washing steps at 5° C. in 3 Mtetramethyl-ammonium chloride, 2 mM EDTA, 0.1% SDS, and 50 mM Tris-HCl(pH 8.0), as described by Wood et al. (1985). At the washing temperatureof 65° C., there was a single hybridization band of ˜2.4 Kb. Thistemperature was chosen for screening a S. cerevisiae genomic phage γEMBL3A DNA library. Screening of the phage EMBL3A library wasessentially the same as described by Winter and Varshavsky (1989).Thirteen positive phage plaques were isolated. Phage DNAs from 11 cloneswere purified from the confluent plate lysates. The DNA was thendigested with several restriction enzymes and a Southern blot wasperformed, as described previously. The restriction pattern andhybridization analysis revealed that the clones fell into three classes.One phage clone with a 3.1 Kb EcoRI and BamHI fragment was chosen forsubcloning into pBluescript. Sequence analysis was carried out asdescribed by Sanger et al. (1977). The 3.1 Kb DNA fragment was sequencedon both strands with synthetic oligonucleotides as primers using the USBSequenase kit (Version 2.0).

[0166] Expression of Zuotin in E. coli

[0167] Zuotin was expressed in E. coli using the T7 pET expressionsystem (Studier et al., 1990). In order to insert ZUO1 into the NcoIsite of the pET8c vector, two bases flanking the initiation ATG weremodified: at −1 (GC) and at +4 (TG). This produced an amino acid changeimmediately after Met of (Phe→Val). An oligonucleotide of 25 bases,CAAGAGTAACCATGGTTTCTTTACC (SEQ ID NO 18), was synthesized and used as aprimer for PCR amplification. A fragment of 1.8 Kb containing the entirecoding region of zuotin was amplified by polymerase chain reaction (PCR)from pSKIIZUO1 and ligated into pET8c which was previously digested withNcoI and BamHI and dephosphorylated. The pETZUO1 clones were isolated bycolony hybridization using the coding region of ZUO1 as a probe.Extensive restriction mapping and sequencing using the T7 primer andseveral internal primers confirmed the correct in-frame cloning of ZUO1.Furthermore, the zuotin expressed in E. coli had its N-terminal regionsequenced and its composition analysed by hydrolysis in order to confirmexpression of the correct protein (Table 4 and FIG. 12B).

[0168]E. coli strain, BL21E3LysS, which carries T7 RNA polymerase in thechromosome of the host under the control of the lacUV5 promoter, wastransformed with pETZUO1. The transformants were induced with 0.5 mMIPTG after the cells had reached a density of 0.5 O.D. (A₆₀₀). Cellswere harvested 3 hours after induction, lysed, treated and analysed asdescribed by Sambrook et al. (1989).

[0169] After cells were lysed by sonication, the cell suspension wascentrifuged at 8000×g for 20 minutes at 4° C., and both supernatant andpellet were saved. The pellet was resuspended in buffer containing 10 mMTris HCl, 50 mM NaCl and 1 mM PMSF, and then urea was added to a finalconcentration of 4 M in order to denature the inclusion bodies. Thesuspension was stirred at 4° C. for 4 hours. The suspension was thendialysed overnight at 4° C. with three changes of buffer. The dialysedsuspension was centrifuged at 10,000×g for 30 minutes. The supernatantwas then loaded on the phosphocellulose column. The column was washed in100 mM KH₂PO₄ buffer and eluted with 1.0 M KH₂PO₄. The eluent wasdialyzed and used for characterization.

[0170] Construction of zuo1 Disruption Mutants

[0171] The gene disruption method of Rothstein (1983) was used forgenerating the zuo1 mutants in the yeast genome. A 1.17 Kb HindIIIfragment containing URA3 was inserted at the unique HindIII site withinthe coding region of ZUO1 (corresponding to amino acid position 186).The DNA was then cut with EcoRI and BamHI, and the released fragment wasused to transform a diploid S. cerevisiae DM27. Standard techniques wereused for yeast transformation, sporulation and tetrad dissection(Sherman et al., 1986). Both orientations of the URA3 insert were usedand yielded the same results.

[0172] Computer Analysis of Zuotin

[0173] The predicted structure of zuotin was analysed by computeralgorithms using Pepplot, FastA, BLAST and others, in the GeneticsComputer Group (GCG) package, as installed at the Whitaker CollegeComputer Facility at the Massachusetts Institute of Technology.GeneWorks Version 2.0 (1991, Intelligenetics, Inc., Mountain View) wasused for zuotin alignment with DnaJ and other proteins. TABLE 1 PeptideSequence^(a) DMEM^(b) PBS Water Structure^(c) EAK16Ac-HN-AEAEAKAAEAEAKAK-CON₂ ++++ ++++ − β EAK12 Ac-HN-AEAKAEEAKAK-CON₂++ + − @, β EAK8 H₂N-AEAEAKAK-COOH − − − RC RAD16H₂N-APAPADADARARADAD-COOH ++++ ++++ − ND ARD8 H₂N-ARAPADAD-COOH − − − NDβ-Amyloid (1-28) H₂N-DAEFRHDSGYEV-HHQKLVFFAEDVGSNK-COOH − − − RC, α, ββ-Amyloid (25-35) H₂N-GSNKGAIIGLM-CONH₂ − − − ND Substance PH₂N-RPKQQFGLM-COHN₂ − − − ND Spantide H₂N-(D)RPKPQQ(D)WL(D)L-CONH₂ − − −ND

[0174] TABLE 2 Potential membrane-forming peptides Name Sequence (N→C)KAE16 AKAKAEAEAKAKAEAE (SEQ ID NO: 32) AKE16 AKAEAKAEAKAEAKAE (SEQ IDNO: 33) EKA16 EAKAEAKAEAKAEAKA (SEQ ID NO: 34) KEAlE KAEAKAEAKAEAKAEA(SEQ ID NO: 35) AEK16 AEAKAEAKAEAKAEAK (SEQ ID NO: 36) DAR16ADADARARADADARAR (SEQ ID NO: 37) RADl6 ARADARADARADARAD (SEQ ID NO: 38)DPA1G DARADARADARADARA (SEQ ID NO: 39) RDA16 RADARADARADARADA (SEQ IDNO: 40) ADR16 ADARADAPADARADAR (SEQ ID NO: 41) ARDAKE16 ARADAKAEARADAKAE(SEQ ID NO: 42) AKEARD16 AKAEARADAKAEARAD (SEQ ID NO: 43) ARKADE16ARAKADAEARAKADAE (SEQ ID NO: 44) AKRAED16 AKARAEADAKARADAE (SEQ ID NO:45) AQ16 AQAQAQAQAQAQAQAQ (SEQ ID NO: 46) VQ16 VQVQVQVQVQVQVQVQ (SEQ IDNO: 47) YQ1G YQYQYQYQYQYQYQYQ (SEQ ID NO: 48) HQ16 HQHQHQHQHQHQHQHQ (SEQID NO: 49) AN16 ANANANANANANANAN (SEQ ID NO: 50) VN16 VNVNVNVNVNVNVNVN(SEQ ID NO: 51) YN16 YNYNYNYNYNYNYNYN (SEQ ID NO: 52) HN16HNHNHNHNHNHNHNHN (SEQ ID NO: 53) ANQiE ANAQANAQANAQANAQ (SEQ ID NO: 54)AQN16 AQANAQANAQANAQAN (SEQ ID NO: 55) VNQ16 VNVQVNVQVNVQVNVQ (SEQ IDNO: 56) VQN16 VQVNVQVNVQVNVQVN (SEQ ID NO: 57) YNQ16 YNYQYNYQYNYQYNYQ(SEQ ID NO: 58) YQN16 YQYNYQYNYQYNYQYN (SEQ ID NO: 59) HNQ16HNHQHNHQHNHQHNHQ (SEQ ID NO: 60) HQN16 HQHNHQHNHQHNHQHN (SEQ ID NO: 61)AKQD18 AKAQADAKAQADAKAQAD (SEQ ID NO: 19) VKQ18 VKVQVDVKVQVDVKVQVD (SEQID NO: 62) YKQ18 YKYQYDYKYQYDYKYQYD (SEQ ID NO: 63) HKQ18HKHQHDHKHQHDHKHQHD (SEQ ID NO: 64)

[0175] TABLE 3 Optical properties of EAK16 at different temperature λ₁ -[θ₁] λ₂ [θ₂] Ratio Temp (° C.) 218 nm X1,000 195 nm X1,000 [θ₂]/[θ₁] 2516 62 3.9 37 15 60 4.0 55 14 55 3.9 70 13 54 4.1 90 12.5 50 4.0

[0176] TABLE 4 Amino acid composition analysis of zuotin HydrolysisDeduced from E. coil (%) Yeast DNA (%) Arg 5.7 7.6 6.0 Lys 9.2 11.9 12.9His 3.1 1.4 1.6 Glu (11.5) (12.1) 9.7 Gln 2.3 Asp (11.0) (11.5) 8.8 Asn3.2 Ser 7.0 7.4 6.9 Thr 4.4 5.0 5.1 Tyr 2.4 1.8 2.1 Ala 11.2 13.0 12.9Val 6.3 4.8 5.5 Leu 6.8 6.6 5.8 Ile 3.6 3.0 3.2 Phe 3.8 4.5 5.1 Trp 0.00.0 1.4 Pro 4.0 3.2 3.5 Gly 5.0 6.1 3.0 Met 0.7 0.1 0.7 Cys 0.0 0.0 0.2

EXAMPLE 6 In Vitro Cell Attachment and Growth

[0177] The suitability of these materials for supporting in vitro cellattachment and growth was tested by introducing a variety of culturedcells to membranes formed by EAK16, RAD16, heteropolymers of EAK16 andRAD16 and RADA16. Cultured cells formed stable attachments andproliferated on the biomaterials. The biomaterials containing the RADsequence provided a better support for the cell types tested (Tables5-7).

[0178] 3T3 fibroblast cells were introduced to membranes of RAD16 andEAK16 in normal serum-containing medium or in serum-free mediumcontaining 30 μg/ml cyclohexamide (cells used for this condition werepretreated in this medium for 2 hours before plating). Cells wereallowed to attach to the membranes for 30 minutes, 1 and 8 hours at 37°C. The attached cells were shaken for 10 minutes on a rotary rocker at60 r.p.m., then scored under phase contrast microscopy. The decrease incells attached to the membrane in the serum-free condition reflects cellloss due to serum deprivation. Similar decreases in cells were observedwith cells plated on tissue culture plastic. The results are reorted inTable 5. TABLE 5 Cell attachment to RAD16 and EAK16 biopolymer sheets isindependent of serum and cell-secreted cell attachment factors Celltype-bio- polymer-attachment medium with serum, serum-free medium, +period no cyclohexamide cyclohexamide 3T3-RAD16 30 min ++++ ++++3T3-RAD16 60 min ++++ ++++ 3T3-RAD16 8 hrs ++++ ++ 3T3-EAK16 30 min ++++++++ 3T3-EAK16 60 min ++++ ++++ 3T3-EAK16 8 hrs ++++ ++

[0179] Cells were pretreated in serum-free medium containing 30 μg/mlcyclohexamide for 2 hours before introduction to the peptide membranes.The cells used for the calcium and magnesium-free condition werepre-equilibrated in Ca⁺⁺ and Mg⁺⁺ free medium containing 5 mM EDTA for15 minutes before introduction to the peptide membranes. Cells wereallowed to attach to the peptide membranes for 30 minutes, 1 and 8 hoursat 37° C. The attached cells were shaken for 10 minutes on a rotaryrocker at 60 r.p.m., then scored under phase contrast microscopy. Theresults are reorted in Table 6. TABLE 6 Cell attachment to RAD16 andEAK16 biopolymer sheets is integrin-independent⁺ occurs in Ca⁺⁺ and Mg⁺⁺free medium Cell type-bio- Normal Ca⁺⁺ & Mg⁺⁺ Ca⁺⁺ & Mg⁺⁺ free, polymerattachment serum-free + EDTA serum-free + period Cyclohex Cyclohex3T3-RAD16 30 min ++++ +++ 3T3-RAD16 30 min ++++ ++++ 3T3-EAK16 30 min++++ ++++ 3T3-EAK16 60 min ++++ ++++ PC12-RAD16 30 min ++++ ++++PC12-RAD16 60 min ++++ ++++ PC12-EAK16 30 min ++++ ++++ PC12-EAK16 60min ++++ ++++ PC12_(NGF)RAD16 30 min ++++ ++++ PC12_(NGF)RAD16 60 min++++ ++++ PC12_(NGF)EAR16 30 min ++++ ++++ PC12_(NGF)EAR16 60 min ++++++++

[0180] The membranes were formed by addition of the oligopeptides to thecell culture media in the presence of 10 μg/ml Congo red. The cells werethen applied to the culture media and allowed attachment to thepeptide-membranes at 37° C. for 2-3 hours. The visible membranes weretransferred to another set of culture media. Cells on membranes wereobserved under a phase contrast microscope every two days for two weeks.TABLE 7 Cells Attached on the Self-Complementary Oligopeptide-MembraneEAK16/ Cell Type Cell Line EAK16 RAD16 RADA16 RAD16 Mouse FibroblastNIH-3T3 +++ ++++ ++ ++ Chick Embryo CEF +++ ++++ ++++ +++ FibroblastHuman Foreskin HFF +++ ++++ ++++ +++ Fibroblast Chinese Hamster CHO +++++++ ++++ +++ Ovary Human Cervix Hela (HLB6) +++ ++++ ++++ +++Fibroblast Human Bone MG63 +++ ++++ ++++ +++ Human Liver HepG2 +++ ++++++++ +++ Hamster HIT-T15 ++ +++ N/D ++ Pancrease Rat Neuron PC12 +++++++ ++++ ++++ Rat Neuron NGF- PC12/NGF +++ ++++ N/D ++++ differentiatedHuman SH-SY5Y +++++ +++++ N/D N/D Neuroblastoma Mouse +++++ +++++ N/DN/D Cerebellum Granule Cells Human ++ +++ N/D N/D Keratinocytes

EXAMPLE 7 In Vitro Neurite Outgrowth

[0181] The suitability of these materials for supporting in vitroneurite outgrowth was tested by treating biomaterial-attached PC12 cellswith nerve growth factor (NGF). This treatment induces PC12 cells todifferentiate into sympathetic neurons which can send out long processescalled neurites. There was a marked difference in the neurite promotingproperties of the two materials tested (EAK16 and RAD16). Nerve growthfactor treated PC12 cells sent out robust neurite processes on the RAD16membranes, while little neurite processes were seen on the EAK16membranes.

[0182] Nerve growth factor differentiated PC12 cells have been usedextensively in studies of neurite outgrowth. PC12 cells upregulate thenumber of calcium-dependent and -independent cell adhesion receptors inresponse to nerve growth factor. Cell attachment and neurite outgrowthfrom nerve growth factor differentiated PC12 cells was examined onmembranes of RAD16 and EAK16 in order to determine whether membranescontaining RGD-like sequences would preferentially support these cellactivities. Neurite outgrowth on peptide membranes is of interest forpotential applications of nerve repair.

[0183] Untreated PC12 cells were introduced to peptide membranes ofEAK16 and RAD16 suspended in 85% RMMP1, 10% heat inactivated horseserum, 5% fetal bovine serum. Half of the cells were treated with nervegrowth factor (50 ng/ml, NGF) and were supplemented with NGF every 3-4days for 14 days. Robust neurite outgrowth was observed from NGF-treatedcells on the tissue culture plastic and the NGF-treated cells on theRAD16 membrane. The neurite outgrowth reached greater than 400 μm inlength, approximately 15-20 times the cell body, following the contourof the matrices in a three dimensional network. Neurite outgrowth wasabsent for all untreated groups and little neurite outgrowth was seen onand NGF-treated EAK16 groups. TABLE 8 Neurite outgrowth from untreatedand nerve growth factor treated PC12 cells on EAK16 and RAD16biopolymers and tissue culture plastic Biopolymer untreated, 14 days NGFtreated, 14 days EAK16 − −1+ RAD16 − ++++

EXAMPLE 8

[0184] In order for a biomaterial to be useful for transplantation, itmust be shown that the material is non-toxic in vivo. Non-antigenicityis another desirable property in order to limit host rejection. Attemptsto raise polyclonal antibodies against EAK16 peptides andEAK16-conjugated proteins in rabbits have been unsuccessful to date.This indicates that this material is non-immunogenic. Experiments havebegun to examine the in vivo response to tissue injections of EAK16 andRAD16 in vivo. Gliosis is a well characterized reaction which occurs inthe brain in response to toxic insult. Imflammation and necrosis occurin other tissues in response to toxic insult. In vivo injections ofEAK16 and RAD16 are believed to be non-toxdic as shown by the absence ofgliosis following injections into brain. Additionally, in vivoinjections of EAK16 and RAD16 into muscle did not elicit a discernableinflammatory response.

[0185] In Vivo Injections of EAK16 and RAD16

[0186] Three hundred g rats were anesthesized with equithesin (0.3ml/100 g body weight). The skull was surgically exposed and small burrholes were stereotaxically drilled at AP=+1.5, ML=+/−3.0 relative tobregma. An injection cannula was placed at a depth of −4.5 in thestriatum (a brain structure). Experimental rats received 1 μl of EAK16or RAD16 injected into the striatum. Control rats received similarinjections of ibotenic acid, a well characterized toxin, as a positivecontorl for gliosis. the experimental rats also received 35 μl injectionof EAK16 or RAD16 into the thigh muscle (n=2 for each group). The ratewere allowed to recover, then sacrificed for tissue harvest. Brainsections were prepared used a freezing microtome and processed for nisslstain which reveals normal cell bodies and gliotic scarring. Controlrats which received brain ibotenic acid injections showed robust glioticscarring around the injection site. No such reaction was observed in therats which received injections of EAK16 or RAD16. The muscle tissue ofrats which received muscle injections of EAK16 or RAD16 did not exhibitdiscernable inflammation or necrosis. These preliminary results indicatethat EAK16 and RAD16 elicit little or no toxicity in vivo.

[0187] In summary, these novel peptide membranes can support cellattachment and growth, as well as specialized functions such as neuriteoutgrowth. The combination of unique physical and cell growth promotingproperties of these biomaterials hold significant promise for theirutility for industrial cell culture and biomedical technologies.

REFERENCES

[0188] Azorin et al., Proc. Nat. Acad. Sci. USA 81:5714-5718 (1984)

[0189] Bairoch, Nucleic Acids Res. 19 supp.:2241-2245 (1991)

[0190] Barrow and Zagorski, Science 253:179-182 (1991)

[0191] Behe and Felsenfeld, Proc. Natl. Acad. Sci. USA 78:1619-1623(1981)

[0192] Bianchi et al., EMBO J. 11:1055-1063 (1992)

[0193] Blaho and Wells, J. Biol. Chem. 262:6082-6088 (1987)

[0194] Blumberg and Silver, Nature 349:627-629 (1991)

[0195] Brack and Orgel, Nature 256:383-387 (1975)

[0196] Bullock et al., Mol. Cell. Biol., 6:3948-3953 (1986)

[0197] Caplan and Douglas, J. Cell Biol. 114:609-621 (1991)

[0198] Celenza and Carlson, Science 233:1175-1180 (1986)

[0199] Chou and Fasman, Annu. Rev. Biochem. 47:251-276 (1978)

[0200] Churchill and Travers, Trends Biochem. Sci. 183:92-97 (1991)

[0201] Erickson, Scientific American, September 1992, pp. 163-164

[0202] Fishel et al., Proc. Natl. Acad. Sci. USA 85:36-40 (1988)

[0203] Gay et al., FEBS Letters 291:87-91 (1991)

[0204] Halverson et al., Biochemistry 29:2639-2644 (1990)

[0205] Hamada et al., Proc. Natl. Acad. Sci. USA 79:6465-6469 (1982)

[0206] Hilbich et al., J. Mol. Biol. 50:149-165 (1991)

[0207] Iqbal and Wisniewski, in: Alzheimer's Disease: The StandardReference, Reisberg (ed.), Free Press, Collier Macmillan Publishers,London, 1983, pp. 48-56

[0208] Jaworski et al., Science 238:773-777 (1987)

[0209] Jones, Genetics 85:23-33 (1977)

[0210] Kirschner et al., Proc. Natl. Acad. Sci. USA 84:6953-6957 (1987)

[0211] Laemmli, Nature 227:680-682 (1970)

[0212] Lafer et al., EMBO J. 4:3655-3660 (1985)

[0213] Lechner and Carbon, Cell 64:717-725 (1991)

[0214] Liberek et al., Proc. Natl. Acad. Sci. USA 85:6632-6636 (1988)

[0215] Lilley, Nature 357:282-283 (1991)

[0216] Liu and Wang, Proc. Natl. Acad. Sci. USA 84:7024-7028 (1987)

[0217] Lizardi, Cell 18:581-589 (1979)

[0218] Luke et al., J. Cell Biol. 114:623-638 (1991)

[0219] Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1982

[0220] Marqusee and Baldwin, Proc. Natl. Acad. Sci. USA 84:8898-8902(1987)

[0221] Marqusee et al., Proc. Natl. Acad. Sci USA 86:5286-5290 (1989)

[0222] McCarthy and Heywood, Nucleic Acids Res. 15:8069-8085 (1987)

[0223] Moller et al., J. Biol. Chem. 257:12081-12085 (1982)

[0224] Moreno and Nurse, Cell 61:5 49-551 (1990)

[0225] Mura and Stollar, Biochemistry 23:6147-6152 (1984)

[0226] Naylor and Clark, Nucleic Acids Res. 18:1595-1601 (1990)

[0227] Nordheim and Rich, Nature 303:674-679 (1983)

[0228] Osterman and Kaiser, J. Cell. Biochem. 29:57-72 (1985)

[0229] Pabo and Sauer, Annu. Rev. Biochem. 53:293-322 (1984)

[0230] Padmanabhan et al., Nature 344:268-270 (1991)

[0231] Pauling, Nature of the Chemical Bond and the Structure ofMolecules and Crystals: An Introduction to Model Structural Chemistry,3rd ed., Cornell University Press, Ithaca, N.Y., 1960

[0232] Pears, Histochemistry, Theoretical and Applied, 2nd Ed., Little,Brown and Company, Boston, 1960

[0233] Peck et al., Proc. Natl. Acad. Sci. USA 79:4560-4564 (1982)

[0234] Piggion et al., Biopolymers 11:633-643 (1972)

[0235] Raabe and Manley, Nucleic Acids Res. 19:6645 (1991)

[0236] Rahmouni and Wells, Science 246:358-363 (1989)

[0237] Rich et al., Annu. Rev. Biochem. 53:791-864 (1984)

[0238] Rippon et al., J. Mol. Biol. 75:369-375 (1973)

[0239] Robbins et al., Cell 64:615-623 (1991)

[0240] Rothstein, Methods Enzymol. 101:202-211 (1983)

[0241] Rott et al., Virology 165:74-86 (1988)

[0242] Russell et al., EMBO J. 2:1647-1653 (1983)

[0243] Sadler et al., J. Cell Biol. 109:2665-2675 (1989)

[0244] Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed.,Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989

[0245] Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)

[0246] Seeman et al., Proc. Natl. Acad. Sci. USA 73:804-808 (1976)

[0247] Seipke et al., Biopolymers 13:1621-1633 (1974)

[0248] Sharp et al., Nucleic Acids Res. 14:5125-5143 (1986)

[0249] Sherman et al., Methods in Yeast Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986

[0250] St. Pierre et al., Biopolymers 17:1837-1847 (1978)

[0251] Studier et al., Methods Enzymol. 185:60-89 (1990)

[0252] Takeuchi et al., FEBS Letters 279:253-255 (1991)

[0253] Treco and Arnheim, Mol. Cell Biol. 6:3943-3947 (1986)

[0254] Trudelle, Polymer 16:9-15 (1975)

[0255] Tsao et al., Cell 56:111-118 (1989)

[0256] Vardimon and Rich, Proc. Natl. Acad. Sci. USA 81:3268-3272 (1983)

[0257] Wahls et al., Mol. Cell Biol. 10:785-793 (1990)

[0258] Winter and Varshavsky, EMBO J. 8:1867-1877 (1989)

[0259] Wittig et al., J. Cell Biol. 108:755-761 (1989)

[0260] Wood et al., Proc. Natl. Acad. Sci. USA 82:1585-1588 (1985)

[0261] Zinsmaier et al., J. Neurogenet. 7:15-29 (1990)

[0262] Zylicz et al., EMBO J. 8:1601-1608

[0263] Equivalents

[0264] Those skilled in the art will know, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

1 64 3083 base pairs nucleic acid double linear DNA (genomic) CDS1292..2590 /product= “zuotin” 1 CCAAAGTGAA TGATATGGGG CTAGAAACGTGTGTTACTTT AGGTATGGTT GATCAAGATC 60 AAGCAAAGCA ATTGAAAGAT GCAGGTTTGACTGCATACAA CCATAACATC GACACTTCCA 120 GAGAACACTA TAGTAAGGTC ATCACCACGAGAACCTACGA CGACAGGTTA CAGACCATCA 180 AGAATGTCCA AGAATCTGGA ATAAAAGCCTGTACCGGTGG TATTTTGGGT CTCGGTGAAA 240 GCGAAGACGA CCATATAGGA TTCATCTACACATTATCCAA TATGTCTCCT CATCCTGAGT 300 CCCTACCAAT TAATAGACTA GTTGCTATCAAAGGGACTCC AATGGCTGAG GAACTTGCCG 360 ATCCAAAGAG TAAAAAGTTG CAATTCGACGAAATTTTGAG AACCATTGCC ACAGCGAGAA 420 TAGTTATGCC AAAGGCCATT ATAAGACTTGCCGCTGGTCG TTATACAATG AAAGAAACAG 480 AGCAATTTGT CTGTTTCATG GCAGGTTGTAACAGTATCTT CACCGGTAAG AAAATGCTGA 540 CGACAATATA TAACGGTTGG GACGAAGACAAGGCAATGTT GGCTAAATGG GGATTGCAAC 600 CTATGGAGGC ATTTAAGTAC GACAGATCTTGAAGATAGGG ATATGTGGAT AATTCTACGA 660 TTCTAACTGT ACATTTCTCC CTTATTTATTAAGAAAACCT ATATATATAT ATATTTACCT 720 ATTTATTCTG CCATCGTTAG CTGGCGTTTTATCTTTTATG CATCCAATAT CTAATATTAC 780 TTCCGATCAC GCATTTAGTT CTGATTACAGCAGAAATCGT AGCGCGATGA GACATTTCAT 840 CAAATGGCCT TTTTTTTTTG GGCAATTTTTTTATATCTTG AAATGATAGT TGCCTTGTAC 900 TTTCAACCGT TCATTTCATT AAGAACTTGACTAAATATGA ACATTTCTTA AAAAAAAAGG 960 TTGACATATA AAAATAATCG AATATAAACGATGGAATTTT TATAAAATTA AACACATATA 1020 TATATATATA TTAACTATAA ATATGTCAAAGAAACCATAC AATCATAGAT TTATAACTAT 1080 CTTTTGGATG ACATTAATGA ACATAACGCTCCTAATACAA ATGTCAAAAA ATATTACCCG 1140 CAAATACGAA TCTTTTTTTT TTCTCGATGAAATTTTGCAA AGAGTTCGAA ATTTTTATTT 1200 CAAGAGCTGG TAGAGAAAAT TTCATAAGGTTTTCCTACCG ATGCTTTTAT AAAATCTTCG 1260 TTTTGTCTCA CATATACCAA CAAGAGTAAC GATG TTT TCT TTA CCT ACC CTA 1312 Met Phe Ser Leu Pro Thr Leu 1 5 ACC TCAGAC ATC ACT GTT GAA GTC AAC AGT TCC GCT ACC AAA ACC CCA 1360 Thr Ser AspIle Thr Val Glu Val Asn Ser Ser Ala Thr Lys Thr Pro 10 15 20 TTC GTC CGTCGT CCG GTC GAA CCG GTT GGT AAG TTC TTT TTG CAA CAT 1408 Phe Val Arg ArgPro Val Glu Pro Val Gly Lys Phe Phe Leu Gln His 25 30 35 GCT CAA AGA ACTTTG AGA AAC CAC ACC TGG TCT GAA TTT GAA AGA ATT 1456 Ala Gln Arg Thr LeuArg Asn His Thr Trp Ser Glu Phe Glu Arg Ile 40 45 50 55 GAA GCT GAA AAGAAC GTC AAA ACC GTT GAT GAA TCC AAT GTC GAC CCA 1504 Glu Ala Glu Lys AsnVal Lys Thr Val Asp Glu Ser Asn Val Asp Pro 60 65 70 GAT GAG TTG TTA TTCGAC ACT GAA TTG GCC GAT GAA GAT TTA CTG ACT 1552 Asp Glu Leu Leu Phe AspThr Glu Leu Ala Asp Glu Asp Leu Leu Thr 75 80 85 CAT GAT GCT AGA GAC TGGAAA ACT GCC GAT TTG TAT GCT GCT ATG GGT 1600 His Asp Ala Arg Asp Trp LysThr Ala Asp Leu Tyr Ala Ala Met Gly 90 95 100 TTG TCT AAG TTG CGT TTCAGA GCT ACT GAA AGT CAA ATC ATC AAG GCT 1648 Leu Ser Lys Leu Arg Phe ArgAla Thr Glu Ser Gln Ile Ile Lys Ala 105 110 115 CAC AGA AAA CAA GTT GTCAAG TAC CAT CCA GAC AAG CAA TCT GCT GCT 1696 His Arg Lys Gln Val Val LysTyr His Pro Asp Lys Gln Ser Ala Ala 120 125 130 135 GGT GGT AGT TTG GACCAA GAT GGC TTT TTC AAG ATT ATT CAA AAG GCC 1744 Gly Gly Ser Leu Asp GlnAsp Gly Phe Phe Lys Ile Ile Gln Lys Ala 140 145 150 TTT GAA ACT TTG ACTGAT TCC AAC AAG AGA GCT CAG TAC GAC TCA TGT 1792 Phe Glu Thr Leu Thr AspSer Asn Lys Arg Ala Gln Tyr Asp Ser Cys 155 160 165 GAT TTT GTT GCC GATGTT CCT CCT CCA AAG AAG GGT ACC GAT TAT GAC 1840 Asp Phe Val Ala Asp ValPro Pro Pro Lys Lys Gly Thr Asp Tyr Asp 170 175 180 TTT TAT GAA GCT TGGGGC CCC GTT TTC GAA GCT GAA GCT CGT TTT TCT 1888 Phe Tyr Glu Ala Trp GlyPro Val Phe Glu Ala Glu Ala Arg Phe Ser 185 190 195 AAG AAG ACT CCT ATTCCT TCT CTA GGT AAC AAA GAT TCT TCC AAG AAG 1936 Lys Lys Thr Pro Ile ProSer Leu Gly Asn Lys Asp Ser Ser Lys Lys 200 205 210 215 GAA GTT GAA CAATTC TAT GCT TTC TGG CAC AGA TTT GAC TCC TGG AGA 1984 Glu Val Glu Gln PheTyr Ala Phe Trp His Arg Phe Asp Ser Trp Arg 220 225 230 ACC TTT GAG TTCTTG GAC GAA GAT GTC CCA GAT GAC TCT TCT AAC AGA 2032 Thr Phe Glu Phe LeuAsp Glu Asp Val Pro Asp Asp Ser Ser Asn Arg 235 240 245 GAC CAC AAG CGTTAC ATT GAA AGA AAG AAC AAG GCC GCA AGA GAC AAG 2080 Asp His Lys Arg TyrIle Glu Arg Lys Asn Lys Ala Ala Arg Asp Lys 250 255 260 AAG AAG ACT GCTGAT AAC GCT AGA TTG GTC AAA CTT GTT GAA AGA GCT 2128 Lys Lys Thr Ala AspAsn Ala Arg Leu Val Lys Leu Val Glu Arg Ala 265 270 275 GTC AGT GAA GATCCC CGT ATC AAA ATG TTC AAA GAA GAA GAG AAG AAG 2176 Val Ser Glu Asp ProArg Ile Lys Met Phe Lys Glu Glu Glu Lys Lys 280 285 290 295 GAA AAG GAAAGA AGA AAA TGG GAA AGA GAA GCC GGT GCC AGA GCT GAA 2224 Glu Lys Glu ArgArg Lys Trp Glu Arg Glu Ala Gly Ala Arg Ala Glu 300 305 310 GCT GAA GCTAAG GCC AAG GCC GAA GCT GAA GCG AAG GCT AAA GCT GAA 2272 Ala Glu Ala LysAla Lys Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu 315 320 325 TCT GAA GCCAAG GCT AAC GCC TCC GCA AAA GCT GAC AAA AAG AAG GCT 2320 Ser Glu Ala LysAla Asn Ala Ser Ala Lys Ala Asp Lys Lys Lys Ala 330 335 340 AAG GAA GCTGCT AAG GCC GCC AAG AAA AAG AAC AAG AGA GCC ATC CGT 2368 Lys Glu Ala AlaLys Ala Ala Lys Lys Lys Asn Lys Arg Ala Ile Arg 345 350 355 AAC TCT GCTAAG GAA GCT GAC TAC TTT GGT GAT GCT GAC AAG GCC ACC 2416 Asn Ser Ala LysGlu Ala Asp Tyr Phe Gly Asp Ala Asp Lys Ala Thr 360 365 370 375 ACG ATTGAC GAA CAA GTT GGT TTG ATC GTT GAC AGT TTG AAT GAC GAA 2464 Thr Ile AspGlu Gln Val Gly Leu Ile Val Asp Ser Leu Asn Asp Glu 380 385 390 GAG TTAGTG TCC ACC GCC GAT AAG ATC AAG GCC AAT GCT GCT GGT GCC 2512 Glu Leu ValSer Thr Ala Asp Lys Ile Lys Ala Asn Ala Ala Gly Ala 395 400 405 AAG GAAGTT TTG AAG GAA TCT GCA AAG ACT ATT GTC GAT TCT GGC AAA 2560 Lys Glu ValLeu Lys Glu Ser Ala Lys Thr Ile Val Asp Ser Gly Lys 410 415 420 CTA CCATCC AGC TTG TTG TCC TAC TTC GTG TGAATACCGT AAGAAATGGA 2610 Leu Pro SerSer Leu Leu Ser Tyr Phe Val 425 430 ATAGAATATA TACGAATGTA TACGAATATTATAGAGAACG TTCTCTTTTA TTTCTATAAT 2670 GAATAGGTTC GGGTAACGGT TCCCTTTTTAGGTATTTCTA GAAGATGAGA GAAGAGGGAA 2730 TAATGAGAAA GGCGAAAAAT AAAGACACCTTTAACGAAAG ATCAAAGGTG TCCTTATTTA 2790 CTTACAATAG CTGCAATTAG TACGACTCAAAAAAAGTGAA AACAAAACTG AAAGGATAGA 2850 TCAATGTCTT ACAGAGGACC TATTGGAAATTTTGGCGGAT AGCCAATGTC ATCATCGCTT 2910 GGACCATACT CTGGCGGTGC ACAATTCCGATCAAACCAGA ACCAATCCAC TTCTGGCATC 2970 TTAAAGCAAT GGAAGCATTC TTTTGAAAAGTTTGCCTCCA GAATTGAGGG GCTCACTGAC 3030 AATGCAGTTG TTTATAAATT GAAGCCTTACATTCCAAGTT TGTCAAGATT TTT 3083 433 amino acids amino acid linear protein2 Met Phe Ser Leu Pro Thr Leu Thr Ser Asp Ile Thr Val Glu Val Asn 1 5 1015 Ser Ser Ala Thr Lys Thr Pro Phe Val Arg Arg Pro Val Glu Pro Val 20 2530 Gly Lys Phe Phe Leu Gln His Ala Gln Arg Thr Leu Arg Asn His Thr 35 4045 Trp Ser Glu Phe Glu Arg Ile Glu Ala Glu Lys Asn Val Lys Thr Val 50 5560 Asp Glu Ser Asn Val Asp Pro Asp Glu Leu Leu Phe Asp Thr Glu Leu 65 7075 80 Ala Asp Glu Asp Leu Leu Thr His Asp Ala Arg Asp Trp Lys Thr Ala 8590 95 Asp Leu Tyr Ala Ala Met Gly Leu Ser Lys Leu Arg Phe Arg Ala Thr100 105 110 Glu Ser Gln Ile Ile Lys Ala His Arg Lys Gln Val Val Lys TyrHis 115 120 125 Pro Asp Lys Gln Ser Ala Ala Gly Gly Ser Leu Asp Gln AspGly Phe 130 135 140 Phe Lys Ile Ile Gln Lys Ala Phe Glu Thr Leu Thr AspSer Asn Lys 145 150 155 160 Arg Ala Gln Tyr Asp Ser Cys Asp Phe Val AlaAsp Val Pro Pro Pro 165 170 175 Lys Lys Gly Thr Asp Tyr Asp Phe Tyr GluAla Trp Gly Pro Val Phe 180 185 190 Glu Ala Glu Ala Arg Phe Ser Lys LysThr Pro Ile Pro Ser Leu Gly 195 200 205 Asn Lys Asp Ser Ser Lys Lys GluVal Glu Gln Phe Tyr Ala Phe Trp 210 215 220 His Arg Phe Asp Ser Trp ArgThr Phe Glu Phe Leu Asp Glu Asp Val 225 230 235 240 Pro Asp Asp Ser SerAsn Arg Asp His Lys Arg Tyr Ile Glu Arg Lys 245 250 255 Asn Lys Ala AlaArg Asp Lys Lys Lys Thr Ala Asp Asn Ala Arg Leu 260 265 270 Val Lys LeuVal Glu Arg Ala Val Ser Glu Asp Pro Arg Ile Lys Met 275 280 285 Phe LysGlu Glu Glu Lys Lys Glu Lys Glu Arg Arg Lys Trp Glu Arg 290 295 300 GluAla Gly Ala Arg Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala 305 310 315320 Glu Ala Lys Ala Lys Ala Glu Ser Glu Ala Lys Ala Asn Ala Ser Ala 325330 335 Lys Ala Asp Lys Lys Lys Ala Lys Glu Ala Ala Lys Ala Ala Lys Lys340 345 350 Lys Asn Lys Arg Ala Ile Arg Asn Ser Ala Lys Glu Ala Asp TyrPhe 355 360 365 Gly Asp Ala Asp Lys Ala Thr Thr Ile Asp Glu Gln Val GlyLeu Ile 370 375 380 Val Asp Ser Leu Asn Asp Glu Glu Leu Val Ser Thr AlaAsp Lys Ile 385 390 395 400 Lys Ala Asn Ala Ala Gly Ala Lys Glu Val LeuLys Glu Ser Ala Lys 405 410 415 Thr Ile Val Asp Ser Gly Lys Leu Pro SerSer Leu Leu Ser Tyr Phe 420 425 430 Val 16 amino acids amino acid linearpeptide 3 Ala Arg Ala Arg Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp AlaAsp 1 5 10 15 28 amino acids amino acid linear peptide 4 Asp Ala Glu PheArg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val PhePhe Ala Glu Asp Val Gly Ser Asn Lys 20 25 11 amino acids amino acidlinear peptide 5 Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met 1 5 10 9amino acids amino acid linear peptide 6 Arg Pro Lys Gln Gln Phe Gly LeuMet 1 5 9 amino acids amino acid linear peptide 7 Arg Pro Lys Pro GlnGln Trp Leu Leu 1 5 52 amino acids amino acid linear protein 8 Ala GluGlu Arg Glu Ile Arg Lys Ala Tyr Lys Arg Leu Ala Met Lys 1 5 10 15 TyrHis Pro Asp Arg Asn Gln Gly Asp Lys Glu Ala Glu Ala Lys Phe 20 25 30 LysGlu Ile Lys Glu Ala Tyr Glu Val Leu Thr Asp Ser Gln Lys Arg 35 40 45 AlaAla Tyr Asp 50 52 amino acids amino acid linear protein 9 Ala Thr GluLys Glu Ile Lys Ser Ala Tyr Arg Gln Leu Ser Lys Lys 1 5 10 15 Tyr HisPro Asp Lys Asn Ala Gly Ser Glu Glu Ala His Gln Lys Phe 20 25 30 Ile GluVal Gly Glu Ala Tyr Asp Val Leu Ser Asp Pro Glu Lys Lys 35 40 45 Lys IleTyr Asp 50 52 amino acids amino acid linear protein 10 Ala Thr Gly AspAsp Ile Lys Lys Thr Tyr Arg Lys Leu Ala Leu Lys 1 5 10 15 Tyr His ProAsp Lys Asn Pro Asp Asn Val Asp Ala Ala Asp Lys Phe 20 25 30 Lys Glu ValAsn Arg Ala His Ser Ile Leu Ser Asp Gln Thr Lys Arg 35 40 45 Asn Ile TyrAsp 50 49 amino acids amino acid linear protein 11 Ala Asn Glu Gln GluLeu Lys Lys Gly Tyr Arg Lys Ala Ala Leu Lys 1 5 10 15 Tyr His Pro AspLys Pro Thr Gly Asp Thr Glu Lys Phe Lys Glu Ile 20 25 30 Ser Glu Ala PheGlu Ile Leu Asn Asp Pro Gln Lys Arg Glu Ile Tyr 35 40 45 Asp 51 aminoacids amino acid linear protein 12 Ala Thr Asp Val Glu Ile Lys Lys AlaTyr Arg Lys Cys Ala Leu Lys 1 5 10 15 Tyr His Pro Asp Lys Asn Pro SerGlu Glu Ala Ala Glu Lys Phe Lys 20 25 30 Glu Ala Ser Ala Ala Tyr Glu IleLeu Ser Asp Pro Glu Lys Arg Asp 35 40 45 Ile Tyr Asp 50 54 amino acidsamino acid linear protein 13 Ala Thr Ala Ala Asp Ile Lys Thr Ala Tyr ArgArg Thr Ala Leu Lys 1 5 10 15 Tyr His Pro Asp Lys Gly Gly Asp Glu GluLys Met Lys Glu Leu Asn 20 25 30 Thr Leu Met Glu Glu Phe Arg Glu Thr GluGly Leu Arg Ala Asp Glu 35 40 45 Thr Leu Glu Asp Ser Asp 50 51 aminoacids amino acid linear protein 14 Ala Ser Asp Arg Asp Ile Lys Ser AlaTyr Arg Lys Leu Ser Val Lys 1 5 10 15 Phe His Pro Asp Lys Leu Ala LysGly Leu Thr Pro Asp Glu Lys Val 20 25 30 Gln Ile Thr Lys Ala Tyr Glu SerLeu Thr Asp Glu Leu Val Arg Gln 35 40 45 Asn Tyr Leu 50 51 amino acidsamino acid linear protein 15 Ala Leu Gly Arg Gly Asp Gln Ala Gly Leu ProPro Pro Gly Leu Arg 1 5 10 15 Tyr His Pro Asp Leu Asn Leu Glu Pro GlyAla Glu Glu Leu Phe Leu 20 25 30 Glu Ile Ala Glu Ala Tyr Asp Val Leu SerAsp Pro Arg Leu Arg Glu 35 40 45 Ile Phe Asp 50 60 amino acids aminoacid linear protein 16 Lys Ala Ala Ala Lys Arg Lys Ala Ala Leu Ala LysLys Lys Ala Ala 1 5 10 15 Ala Ala Lys Arg Lys Ala Ala Ala Lys Ala LysLys Ala Lys Lys Pro 20 25 30 Lys Lys Lys Ala Ala Lys Lys Ala Lys Lys ProAla Lys Lys Ser Pro 35 40 45 Lys Lys Ala Lys Lys Pro Ala Lys Lys Ser ProLys 50 55 60 61 amino acids amino acid linear protein 17 Lys Glu Lys AlaPro Arg Lys Arg Ala Thr Ala Ala Lys Pro Lys Lys 1 5 10 15 Pro Ala AlaLys Lys Pro Ala Ala Ala Ala Lys Lys Pro Lys Lys Ala 20 25 30 Ala Ala ValLys Lys Ser Pro Lys Lys Ala Lys Lys Pro Ala Ala Ala 35 40 45 Ala Thr LysLys Ala Ala Lys Ser Pro Lys Lys Ala Ala 50 55 60 25 base pairs nucleicacid single linear 18 CAAGAGTAAC CATGGTTTCT TTACC 25 18 amino acidsamino acid linear peptide 19 Ala Lys Ala Gln Ala Asp Ala Lys Ala Gln AlaAsp Ala Lys Ala Gln 1 5 10 15 Ala Asp 16 amino acids amino acid linearpeptide 20 Val Arg Val Arg Val Asp Val Asp Val Arg Val Arg Val Asp ValAsp 1 5 10 15 16 amino acids amino acid linear peptide 21 Ala Asp AlaAsp Ala Lys Ala Lys Ala Asp Ala Asp Ala Lys Ala Lys 1 5 10 15 32 basepairs nucleic acid single linear 22 ATGTTTTCTT TGCCAACTTT GACTTCTGAT AT32 11 amino acids amino acid linear peptide 23 Met Val Ser Leu Pro ThrLeu Thr Ser Asp Ile 1 5 10 12 amino acids amino acid linear peptide 24Ala Glu Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys 1 5 10 16 amino acidsamino acid linear peptide 25 Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala LysAla Lys Ala Lys Ala 1 5 10 15 16 amino acids amino acid linear peptide26 Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala 1 510 15 16 amino acids amino acid linear peptide 27 Ala Asp Ala Asp AlaAsp Ala Asp Ala Asp Ala Asp Ala Asp Ala Asp 1 5 10 15 56 amino acidsamino acid linear protein negatively charged amino acid 3, 5, 28, 42, 48non-conserved amino acid 4, 11, 24, 25, 26, 27, 30, 32, 35, 38, 39, 49,50, 53 non-polar amino acid 14, 37, 43, 54 28 Ala Thr Xaa Xaa Xaa IleLys Lys Ala Tyr Xaa Arg Lys Xaa Ala Leu 1 5 10 15 Lys Tyr His Pro AspLys Asn Xaa Xaa Xaa Xaa Xaa Ala Xaa Glu Xaa 20 25 30 Lys Phe Xaa Glu XaaXaa Xaa Ala Tyr Xaa Xaa Leu Ser Asp Pro Xaa 35 40 45 Xaa Xaa Lys Arg XaaXaa Tyr Asp 50 55 5 amino acids amino acid linear peptide 29 Lys Ala LysAla Lys 1 5 5 amino acids amino acid linear peptide 30 Lys Ala His AlaLys 1 5 5 amino acids amino acid linear peptide any amino acid 5 31 LysAla Lys Ala Xaa 1 5 16 amino acids amino acid linear peptide 32 Ala LysAla Lys Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu 1 5 10 15 16amino acids amino acid linear peptide 33 Ala Lys Ala Glu Ala Lys Ala GluAla Lys Ala Glu Ala Lys Ala Glu 1 5 10 15 16 amino acids amino acidlinear peptide 34 Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala GluAla Lys Ala 1 5 10 15 16 amino acids amino acid linear peptide 35 LysAla Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala Lys Ala Glu Ala 1 5 10 15 16amino acids amino acid linear peptide 36 Ala Glu Ala Lys Ala Glu Ala LysAla Glu Ala Lys Ala Glu Ala Lys 1 5 10 15 16 amino acids amino acidlinear peptide 37 Ala Asp Ala Asp Ala Arg Ala Arg Ala Asp Ala Asp AlaArg Ala Arg 1 5 10 15 16 amino acids amino acid linear peptide 38 AlaArg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp 1 5 10 15 16amino acids amino acid linear peptide 39 Asp Ala Arg Ala Asp Ala Arg AlaAsp Ala Arg Ala Asp Ala Arg Ala 1 5 10 15 16 amino acids amino acidlinear peptide 40 Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala ArgAla Asp Ala 1 5 10 15 16 amino acids amino acid linear 41 Ala Asp AlaArg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg 1 5 10 15 16 aminoacids amino acid linear peptide 42 Ala Arg Ala Asp Ala Lys Ala Glu AlaArg Ala Asp Ala Lys Ala Glu 1 5 10 15 16 amino acids amino acid linearpeptide 43 Ala Lys Ala Glu Ala Arg Ala Asp Ala Lys Ala Glu Ala Arg AlaAsp 1 5 10 15 16 amino acids amino acid linear peptide 44 Ala Arg AlaLys Ala Asp Ala Glu Ala Arg Ala Lys Ala Asp Ala Glu 1 5 10 15 16 aminoacids amino acid linear peptide 45 Ala Lys Ala Arg Ala Glu Ala Asp AlaLys Ala Arg Ala Asp Ala Glu 1 5 10 15 16 amino acids amino acid linearpeptide 46 Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln AlaGln 1 5 10 15 16 amino acids amino acid linear peptide 47 Val Gln ValGln Val Gln Val Gln Val Gln Val Gln Val Gln Val Gln 1 5 10 15 16 aminoacids amino acid linear peptide 48 Tyr Gln Tyr Gln Tyr Gln Tyr Gln TyrGln Tyr Gln Tyr Gln Tyr Gln 1 5 10 15 16 amino acids amino acid linearpeptide 49 His Gln His Gln His Gln His Gln His Gln His Gln His Gln HisGln 1 5 10 15 16 amino acids amino acid linear peptide 50 Ala Asn AlaAsn Ala Asn Ala Asn Ala Asn Ala Asn Ala Asn Ala Asn 1 5 10 15 16 aminoacids amino acid linear peptide 51 Val Asn Val Asn Val Asn Val Asn ValAsn Val Asn Val Asn Val Asn 1 5 10 15 16 amino acids amino acid linearpeptide 52 Tyr Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn Tyr Asn TyrAsn 1 5 10 15 16 amino acids amino acid linear peptide 53 His Asn HisAsn His Asn His Asn His Asn His Asn His Asn His Asn 1 5 10 15 16 aminoacids amino acid linear peptide 54 Ala Asn Ala Gln Ala Asn Ala Gln AlaAsn Ala Gln Ala Asn Ala Gln 1 5 10 15 16 amino acids amino acid linearpeptide 55 Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln Ala Asn Ala Gln AlaAsn 1 5 10 15 16 amino acids amino acid linear peptide 56 Val Asn ValGln Val Asn Val Gln Val Asn Val Gln Val Asn Val Gln 1 5 10 15 16 aminoacids amino acid linear peptide 57 Val Gln Val Asn Val Gln Val Asn ValGln Val Asn Val Gln Val Asn 1 5 10 15 16 amino acids amino acid linearpeptide 58 Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn TyrGln 1 5 10 15 16 amino acids amino acid linear peptide 59 Tyr Gln TyrAsn Tyr Gln Tyr Asn Tyr Gln Tyr Asn Tyr Gln Tyr Asn 1 5 10 15 16 aminoacids amino acid linear peptide 60 His Asn His Gln His Asn His Gln HisAsn His Gln His Asn His Gln 1 5 10 15 16 amino acids amino acid linearpeptide 61 His Gln His Asn His Gln His Asn His Gln His Asn His Gln HisAsn 1 5 10 15 18 amino acids amino acid linear peptide 62 Val Lys ValGln Val Asp Val Lys Val Gln Val Asp Val Lys Val Gln 1 5 10 15 Val Asp 18amino acids amino acid linear peptide 63 Tyr Lys Tyr Gln Tyr Asp Tyr LysTyr Gln Tyr Asp Tyr Lys Tyr Gln 1 5 10 15 Tyr Asp 18 amino acids aminoacid linear peptide 64 His Lys His Gln His Asp His Lys His Gln His AspHis Lys His Gln 1 5 10 15 His Asp

1. A method for in vitro cell culture comprising: (a) adding amacroscopic membrane which is formed by self-assembly of amphiphilicpeptides in an aqueous solution containing monovalent metal cations to acell culture medium comprising cells, thereby forming a membrane/culturemixture; (b) maintaining the mixture under conditions sufficient forcell growth.
 2. The method of claim 1 wherein the peptides are greaterthan 12 amino acids in length, have alternating hydrophobic andhydrophilic amino acids, and are complementary and structurallycompatible.
 3. The method of claim 2 wherein the hydrophilic amino acidsare selected from amino acids which can form ionized pairs and aminoacids which can form hydrogen bonds.
 4. The method of claim 1 whereinthe peptides are homogeneous.
 5. The method of claim 1 wherein themacroscopic membrane was formed by self-assembly of a peptide having thesequence (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_(n), where n is greater thanor equal to
 2. 6. The method of claim 1 wherein the macroscopic membraneformed by a peptide having the sequence(Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala)_(n), where n is greater than or equalto
 2. 7. The method of claim 1 wherein the macroscopic membrane is notsubstantially affected by a condition selected from the group consistingof: a) aqueous solution; b) serum; c) ethanol; d) dilution of thepeptide; e) concentration of the monovalent metal cations; f)temperature up to 90° C.; g) pH from 1 to about 11; h) up to 10% sodiumdodecyl sulfate; i) up to 7 M guanidine hydrochloride; j) up to 8 Murea; and k) active protease.
 8. The method of claim 7 wherein theaqueous solution is selected from water, a salt solution, and tissueculture medium.
 9. The method of claim 7 wherein the protease istrypsin, α-chymotrypsin, papain, protease K or pronase.
 10. The methodof claim 1 wherein the cells are mammalian cells.
 11. The method ofclaim 1 wherein the cells are human cells.
 12. A method for forming amacroscopic membrane comprising combining peptides, which are greaterthan 12 amino acids in length, have alternating nonpolar and hydrophilicamino acids, and are complementary and structurally compatible, withmonovalent metal cations in an aqueous solution under conditionssuitable for self-assembly of the peptide into the macroscopic membrane.13. The method of claim 12 wherein the peptides are homogeneous.
 14. Themethod of claim 13 wherein the peptides have a sequence characterized by(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_(n), where n is bigger than or equalto
 2. 15. The method of claim 13 wherein the peptides have a sequencecharacterized by (Ala-Arg-Ala-Arg-Ala-Asp-Ala-Asp)_(n), where n isbigger than or equal to
 2. 16. The method of claim 12 wherein thepeptide is chemically synthesized.
 17. The method of claim 12 whereinthe monovalent metal cations are selected from Li⁺, Na⁺, and K⁺.
 18. Themethod of claim 12 wherein the peptides are added to an aqueous solutioncontaining the monovalent metal cations.
 19. The method of claim 18wherein the aqueous solution is phosphate-buffered saline.
 20. Themethod of claim 12 wherein the suitable conditions comprise the absenceof an inhibitor of the self-assembly of the peptides into themacroscopic membrane.
 21. The method of claim 20 wherein the inhibitoris a divalent metal cation.
 22. The method of claim 20 wherein theinhibitor is sodium dodecyl sulfate.
 23. The method of claim 12 whereinthe suitable conditions comprise a pH of less than
 12. 24. A method forslow-diffusion delivery of a drug comprising administering the drug in avehicle comprising a macroscopic membrane formed by self-assembly ofamphiphilic peptides.
 25. The method of claim 24 wherein the peptideshave a sequence characterized by (Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)_(n),where n is greater than or equal to
 2. 26. The method of claim 24wherein the peptides have a sequence characterized by(Ala-Arg-Ala-Arg-Ala-Asp-Ala-Asp)_(n), where n is greater than or equalto
 2. 27. The method of claim 24 wherein the drug is administeredorally.
 28. A method for identifying a drug which inhibits theself-assembly of amphiphilic peptides into a macroscopic membranecomprising: a) combining a drug with the amphiphilic peptides andmonovalent metal cations in an aqueous solution under conditions whichwould be suitable for self-assembly of the amphiphilic peptides into amacroscopic membrane in the absence of the drug; and b) detectingdecreased membrane formation, wherein decreased membrane formation inthe presence of the drug indicates that the drug inhibits theself-assembly of the amphiphilic peptides into a macroscopic membrane.29. A drug identified by the method of claim
 28. 30. Substantially pureDNA having all or a portion of the nucleotide sequence of FIG. 12C (SEQID NO: 1).
 31. Substantially pure DNA which encodes a protein having allor a biologically active portion of the amino acid sequence of FIG. 1(SEQ ID NO: 2).
 32. Isolated protein having all or a biologically activeportion of the amino acid sequence of FIG. 1 (SEQ ID NO: 2).
 33. Areagent comprising the isolated protein of claim 32, which is asubstrate for a protein kinase selected from the group consisting of: a)CDC28; b) casein kinase II; c) cAMP-dependent protein kinase; d)tyrosine kinase; and e) protein kinase C.
 34. A reagent comprising theisolated protein of claim 32, which binds left-handed Z-DNA.
 35. Thereagent of claim 34 wherein the protein has a portion of the amino acidsequence of FIG. 1 (SEQ ID NO: 2) from amino acids 306 to
 339. 36. Areagent which converts B-DNA to Z-DNA comprising a peptide selected fromthe group consisting of: a) KAKAK (SEQ ID NO: 29); b) KAK; and c) KAHAK(SEQ ID NO: 30).
 37. A reagent which converts B-DNA to Z-DNA comprisingthe peptide KAKAX (SEQ ID NO: 31), where X is any amino acid.